Preparation of deallergenized proteins and permuteins

ABSTRACT

Modified proteins are disclosed that maintain enzymatic and insecticidal activity while displaying reduced or eliminated allergenicity. Epitopes which bind to anti-patatin antibodies were identified, and removed via site directed mutagenesis. Tyrosines were observed to generally contribute to the allergenic properties of patatin proteins. Removal of glycosylation sites was observed to reduce or eliminate antibody binding.  
     Permuteins are also disclosed which have a rearranged amino acid sequence while retaining enzymatic activity.  
     Deallergenized proteins and permuteins can be used as insecticidal materials, as nutritional supplements, and as immunotherapeutic agents.

FIELD OF THE INVENTION

[0001] The invention relates generally to non-naturally occurring novelproteins which have insecticidal properties, and more specifically tothe design, preparation, and use of proteins that have beendeallergenized while maintaining their insecticidal properties.Deallergenized patatin proteins include variants that have hadallergenic sequences modified, and permuteins that have had their aminoacid sequences rearranged at one or more breakpoints.

BACKGROUND OF THE INVENTION

[0002] Insecticidal Proteins

[0003] The use of natural products, including proteins, is a well knownmethod of controlling many insect, fungal, viral, bacterial, andnematode pathogens. For example, endotoxins of Bacillus thuringiensis(B.t.) are used to control both lepidopteran and coleopteran insectpests. Genes producing these endotoxins have been introduced into andexpressed by various plants, including cotton, tobacco, and tomato.There are, however, several economically important insect pests such asboll weevil (BWV), Anthonomus grandis, and corn rootworm (CRW),Diabrotica spp. that are not as susceptible to B.t. endotoxins as areinsects such as lepidopterans. In addition, having other, different geneproducts for control of insects which are susceptible to B.t. endotoxinsis important, if not vital, for resistance management.

[0004] It has been recently discovered that the major storage protein ofpotato tubers, patatins (Gaillaird, T., Biochem. J. 121: 379-390, 1971;Racusen, D., Can. J Bot., 62: 1640-1644, 1984; Andrews, D. L., et al.,Biochem. J, 252: 199-206, 1988), will control various insects, includingwestern rootworm (WCRW, Diabrotica virigifera), southern corn rootworm(SCRW, Diabrotica undecimpunctata), and boll weevil (BWV, Anthonomusgrandis) (U.S. Pat. No. 5,743,477). Patatins are lethal to some larvaeand will stunt the growth of survivors so that maturation is preventedor severely delayed, resulting in no reproduction. These proteins, havenonspecific lipid acyl hydrolase activity and studies have shown thatthe enzyme activity is essential for its insecticidal activity(Strickland, J. A., et al., Plant Physiol., 109: 667-674, 1995; U.S.Pat. No. 5,743,477). Patatins can be applied directly to the plants orintroduced in other ways well known in the art, such as through theapplication of plant-colonizing microorganisms, which have beentransformed to produce the enzymes, or by the plants themselves aftersimilar transformation.

[0005] In potato, the patatins are found predominantly in tubers, butalso at much lower levels in other plant organs (Hofgen, R. andWillmitzer, L., Plant Science, 66: 221-230, 1990). Genes that encodepatatins have been previously isolated by Mignery, G. A., et al.(Nucleic Acids Research, 12: 7987-8000, 1984; Mignery, G. A., et al.,Gene, 62: 27-44, 1988; Stiekema, et al., Plant Mol. Biol., 11: 255-269,1988) and others. Patatins are found in other plants, particularlysolanaceous species (Ganal, et al., Mol. Gen. Genetics, 225: 501-509,1991; Vancanneyt, et al., Plant Cell, 1: 533-540, 1989) and recently Zeamays (WO 96/37615). Rosahl, et al. (EMBO J., 6: 1155-1159, 1987)transferred it to tobacco plants, and observed expression of patatin,demonstrating that the patatin genes can be heterologously expressed byplants.

[0006] Patatin is an attractive for use in planta as an insect controlagent, but unfortunately a small segment of the population displaysallergic reactions to patatin proteins, and in particular to potatopatatin, as described below.

[0007] Food Allergens

[0008] There are a variety of proteins that cause allergic reactions.Proteins that have been identified as causing an allergic reaction inhypersensitive patients occur in many plant and animal derived foods,pollens, fungal spores, insect venoms, insect feces, and animal danderand urine (King, H. C., Ear Nose Throat J., 73(4): 237-241, 1994;Astwood, J. D., et al., Clin. Exp. Allergy, 25: 66-72, 1995; Astwood, J.D. and Fuchs R. L., Monographs in allergy Vol. 32: Highlights in foodallergy, pp. 105-120, 1996; Metcalfe, D. D., et al., Critical Reviews inFood Science and Nutrition, 36S: 165-186, 1996). The offending proteinsof many major sources of allergens have been characterized by clinicaland molecular methods. The functions of allergenic proteins in vivo arediverse, ranging from enzymes to regulators of the cell cytoskeleton.

[0009] To understand the molecular basis of allergic disease, theimportant IgE binding epitopes of many allergen proteins have beenmapped (Elsayed, S. and Apold, J., Allergy 38(7): 449-459, 1983;Elsayed, S. et al., Scand J. Clin. Lab. Invest. Suppl. 204: 17-31 1991;Zhang, L., et al., Mol. Immunol. 29(11): 1383-1389, 1992). The optimalpeptide length for IgE binding has been reported to be between 8 and 12amino acids. Conservation of epitope sequences is observed in homologousallergens of disparate species (Astwood, J. D., et al., Clin. Exp.Allergy, 25: 66-72, 1995). Indeed, conservative substitutions introducedby site-directed mutagenesis reduce IgE binding of known epitopes whenpresented as peptides.

[0010] Food allergy occurs in 2-6% of the population. Eight foods orfood groups (milk, eggs, fish, crustacea, wheat, peanuts, soybeans, andtree nuts) account for 90% of allergies to foods. Nevertheless, over 160different foods have been reported to cause adverse reactions, includingpotato (Hefle, S., et al., Crit. Rev. in Food Sci. Nutr., 36S: 69-90,1996).

[0011] Mode of Action of Allergens

[0012] Regardless of the identity of the allergen, it is theorized thatthe underlying mechanism of allergen response is the same. Immediatehypersensitivity (or anaphylactic response) is a form of allergicreaction which develops very quickly, i.e., within seconds or minutes ofexposure of the patient to the causative allergen, and is mediated by Blymphocyte IgE antibody produciton. Allergic patients exhibit elevatedlevels of IgE, mediating hypersensitivity by priming mast cells whichare abundant in the skin, lymphoid organs, in the membranes of the eye,nose and mouth, and in the respiratory tree and intestines. The IgE inallergy-suffering patients becomes bound to the IgE receptors of mastcells. When this bound IgE is subsequently contacted by the appropriateallergen, the mast cell is caused to degranulate and release varioussubstances such as histamine into the surrounding tissue (Church et al.In: Kay, A. B. ed., Allergy and Allergic Diseases, Oxford, BlackwellScience, pp. 149-197, 1997).

[0013] It is the release of these substances which is responsible forthe clinical symptoms typical of immediate hypersensitivity, namelycontraction of smooth muscle in the airways or in the intestine, thedilation of small blood vessels, and the increase in their permeabilityto water and plasma proteins, the secretion of thick sticky mucus, and(in the skin) the stimulation of nerve endings that result in itching orpain. Immediate hypersensitivity is, at best, a nuisance to the suffer;at worst it can present very serious problems and can in rare cases evenresult in death.

[0014] Allergic Reactions to Potato

[0015] Food allergy to potato is considered rare in the generalpopulation (Castells, M. C., et al., Allergy Clin. Immunol., 8:1110-1114, 1986; Hannuksela, M., et al., Contact Dermatitis, 3: 79-84,1977; Golbert, T. M., et al., Journal of Allergy, 44: 96-107, 1969).Approximately 200 individuals have participated in published clinicalaccounts of potato allergy (Hefle, S. et al., Critical Reviews in FoodScience and Nutrition, 36S: 69-90, 1996). A number of IgE bindingproteins have been identified in potato tuber extracts (see Table 1),however the amino acid sequence and function of these proteins has notbeen determined (Wahl, R., et al., Intl. Arch. Allergy Appl. Immunol.,92: 168-174, 1990). TABLE 1 Studies of potato tuber IgE-binding proteins(allergens) Study Protein Characteristics (Castells, M. C. et al. J.Allergy Clin. Unknown 14 to 40 kDa Immunol. 78, 1110-1114, 1986) (Wahl,R. et al. Int. Arch. Allergy Appl. Unknown 42/43 kDa Immunol. 92:168-174, 1990) Unknown 65 kDa Unknown 26 kDa Unknown 20 kDa Unknown 14kDa Unknown <14 kDa (˜5 kDa) (Ebner, C. et al. in: Wuthrich, B. &Unknown 42/43 kDa Ortolani, C. (eds.), Highlights in food allergy.Monographs in Allergy, Volume 32 Basil, Karger, pp. 73-77, 1996) Unknown23 kDa Unknown ˜16 kDa Unknown <14 kDa (˜5 kDa)

[0016] Improved Safety from the use of Hypoallergenic Proteins

[0017] Patatin has been identified as an allergenic protein (Seppala, U.et al., J. Allergy Clin. Immunol. 103:165-171, 1999). Accordingly,potato allergic subjects may not be able to safely consume productscontaining unmodified patatin protein, such as crops to which foliarapplications of patatins have been applied, or crops which have beenengineered to express patatin. In addition, proliferation of foodallergens in the food supply is considered hazardous (Metcalfe, D. D.,et al., Critical Reviews and Food Science and Nutrition, 36S: 165-186,1996). There are additional concerns regarding the use of potentiallyallergenic food proteins where workers might be exposed to airborneparticulates, initiating a new allergic response (Moneret-Vautrin, D.A., et al., Rev. Med. Interne., 17(7): 551-557, 1996).

[0018] Permuteins

[0019] Novel proteins generated by the method of sequence transpositionresembles that of naturally occurring pairs of proteins that are relatedby linear reorganization of their amino acid sequences (Cunningham, etal. Proc. Natl. Sci., U.S.A., 76: 3218-3222, 1979; Teather, et al., J.Bacteriol., 172: 3837-3841, 1990; Schimming, et al., Eur. J. Biochem.,204: 13-19, 1992; Yamiuchi, et al., FEBS Lett., 260: 127-130, 1991;MacGregor, et al., FEBS. Lett., 378: 263-266, 1996). The first in vitroapplication of sequence rearrangement to proteins was described byGoldenberg and Creighton (Goldenberg and Creighton, J. Mol. Biol., 165:407-413, 1983). A new N-terminus is selected at an internal site(breakpoint) of the original sequence, the new sequence having the sameorder of amino acids as the original from the breakpoint until itreaches an amino acid that is at or near the original C-terminus. Atthis point the new sequence is joined, either directly or through anadditional portion or sequence (linker), to an amino acid that is at ornear the original N-terminus, and the new sequence continues with thesame sequence as the original until it reaches a point that is at ornear the amino acid that was N-terminal to the breakpoint site of theoriginal sequence, this residue forming the new C-terminus of the chain.This approach has been applied to proteins which range in size from 58to 462 amino acids and represent a broad range of structural classes(Goldenberg and Creighton, J. Mol. Biol., 165: 407-413, 1983; Li andCoffino, Mol. Cell. Biol., 13: 2377-2383, 1993; Zhang, et al., NatureStruct. Biol., 1: 434-438, 1995; Buchwalder, et al., Biochemistry, 31:1621-1630, 1994; Protasova, et al., Prot. Eng., 7: 1373-1377, 1995;Mullins, et al., J. Am. Chem. Soc., 116: 5529-5533, 1994; Garrett, etal., Protein Science, 5: 204-211, 1996; Hahn, et al., Proc. Natl. Acad.Sci. USA., 91: 10417-10421, 1994; Yang and Schachman, Proc. Natl. Acad.Sci. U.S.A., 90: 11980-11984, 1993; Luger, et al., Science, 243:206-210, 1989; Luger, et al., Prot. Eng., 3: 249-258, 1990; Lin, et al.,Protein Science, 4: 159-166, 1995; Vignais, et al., Protein Science, 4:994-1000, 1995; Ritco-Vonsovici, et al., Biochemistry, 34: 16543-16551,1995; Horlick, et al., Protein Eng., 5: 427-431, 1992; Kreitman, et al.,Cytokine, 7: 311-318, 1995; Viguera, et al., Mol. Biol., 247: 670-681,1995; Koebnik and Kramer, J. Mol. Biol., 250: 617-626, 1995; Kreitman,et al., Proc. Natl. Acad. Sci., 91: 6889-6893, 1994).

[0020] There exists a need for the development of plant expressibleinsecticidal proteins which possess minimal or no allergenic properties.

SUMMARY OF THE INVENTION

[0021] Novel protein sequences, and nucleic acid sequences encoding themare disclosed. The proteins maintain desirable enzymatic andinsecticidal properties while displaying reduced or eliminatedallergenicity.

[0022] Allergenic epitopes are identified by scanning overlappingpeptide sequences with an immunoreactivity assay. Alanine scanning and‘rational substitution’ is performed on identified peptide sequences todetermine specific amino acids which contribute to antibody binding, andpresumably, to the allergenic properties of the whole protein.Individual mutations are introduced into the whole protein sequence bymethods such as site directed mutagenesis of the encoding nucleic acidsequence to delete or modify the allergenic sequences.

[0023] Glycosylation target residues are identified within amino acidsequences of proteins which have demonstrated allergy elicitingproperties. Glycosylation target amino acid residues are rationallysubstutited with other amino acid residues to eliminate glycosylationand to provide a variant deglycosylated protein. The variant protein maythen exhibit reduced allergen eliciting properties and may also exhibitreduced binding to IgE within serum of patients observed to be allergicto said glycosylated protein.

[0024] Permuteins of the deallergenized protein sequences can beconstructed to further reduce or eliminate allergic reactions. Theencoding nucleic acid sequence is modified to produce a non-naturallyoccurring protein having a linear amino acid sequence different from thenaturally occurring protein sequence, while maintaining enzymatic andinsecticidal properties. The permutein is preferably produced in plantcells, and more preferably produced at a concentration which is toxic toinsects ingesting the plant cells.

[0025] Methods for reducing, eliminating, or decreasing allergeneliciting properties of a protein are specifically contemplated herein.Such methods comprise steps including identifying one or more patientsexhibiting an allergic sensitivity to an allergen eliciting protein andobtaining a sample of serum from the patient; exposing the patient serumto a first set of synthetic overlapping peptides which represent theallergen eliciting protein in order to identify such peptides whichexhibit epitopes which bind to IgE present within the allergic patients'serum and wherein the IgE present in the serum has a specific affinityfor the said allergen eliciting protein; producing a second set ofpeptides which are variant peptides based on the first set of peptideswhich were identified to bind specifically to IgE present in patientserum, wherein the second set variant peptides exhibit alanine scanningor rational scanning amino acid substitutions which exhibit reduced,decreased, or eliminated IgE binding when compared to the first setnon-variant peptides, and wherein such substitutions which reduce,eliminate or decrease IgE binding are identified as result effectivesubstitutions; and modifying the amino acid sequence of the allergeneliciting protein to contain one or more of said result effectivesubstitutions, wherein the modified protein is a variant of the allergeneliciting protein which lacks allergen eliciting protein or exhibitsreduced allergen eliciting properties, and wherein the variant of theallergen eliciting protein comprising one or more result effectivesubstitutions exhibits reduced, decreased, or totally eliminated bindingof IgE present within said patients' serum.

[0026] The novel proteins can be used in controlling insects, asnutritional supplements, in immunotherapy protocols, and in otherpotential applications. Transgenic plant cells and plants containing theencoding nucleic acid sequence can be particularly beneficial in thecontrol of insects, and as a nutritional/immunotherapy material.

DESCRIPTION OF THE FIGURES

[0027] The following figures form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention can be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0028]FIG. 1 illustrates the alignment of potato patatin PatA (acyllipid hydrolase) with patatin (acyl lipid hydrolase) homologs andrelated amino acid sequences, the homologs and related sequences beingfrom both dicot and monocot plant species.

[0029]FIG. 2 illustrates IgE binding to overlapping peptide sequences.

[0030]FIG. 3 illustrates construction of nucleic acid sequences encodingpatatin permutein proteins, and in this figure for illustrative purposesa breakpoint at position 247 is shown.

DESCRIPTION OF THE SEQUENCE LISTINGS

[0031] The following description of the sequence listing forms part ofthe present specification and is included to further demonstrate certainaspects of the present invention. The invention can be better understoodby reference to one or more of these sequences in combination with thedetailed description of specific embodiments presented herein. SEQ IDNO: 1 DNA sequence encoding a patatin (acyl lipid hydrolase) protein SEQID NO: 2 potato patatin protein sequence SEQ ID NO: 3 thermalamplification primer SEQ ID NO: 4 thermal amplification primer SEQ IDNO: 5 thermal amplification product SEQ ID NO: 6 Pre-cleavage patatinprotein produced in Pichia pastoris SEQ ID NO: 7 Post-cleavage patatinprotein produced in Pichia pastoris SEQ ID NO: 8 Y106F mutagenic primerSEQ ID NO: 9 Y129F mutagenic primer SEQ ID NO: 10 Y185F mutagenic primerSEQ ID NO: 11 Y193F mutagenic primer SEQ ID NO: 12 Y185F and Y193Fmutagenic primer SEQ ID NO: 13 Y270F mutagenic primer SEQ ID NO: 14Y316F mutagenic primer SEQ ID NO: 15 Y362F mutagenic primer SEQ ID NO:16-104 Peptide scan sequences of a patatin protein SEQ ID NO: 105-241Alanine and rational scan sequences of selected patatin peptides SEQ IDNO: 242 thermal amplification primer 27 SEQ ID NO: 243 thermalamplification primer 48 SEQ ID NO: 244 thermal amplification primer 47SEQ ID NO: 245 thermal amplification primer 36 SEQ ID NO: 246 pMON37402sequence encoding permutein protein SEQ ID NO: 247 Permutein proteinencoded from pMON37402 sequence SEQ ID NO: 248 thermal amplificationprimer 58 SEQ ID NO: 249 thermal amplification primer 59 SEQ ID NO: 250pMON37405 sequence encoding permutein protein SEQ ID NO: 251 Permuteinprotein encoded by pMON37405 sequence SEQ ID NO: 252 thermalamplification primer 60 SEQ ID NO: 253 thermal amplification primer 61SEQ ID NO: 254 pMON37406 sequence encoding permutein protein SEQ ID NO:255 Permutein protein encoded by pMON37406 sequence SEQ ID NO: 256thermal amplification primer 62 SEQ ID NO: 257 thermal amplificationprimer 63 SEQ ID NO: 258 pMON37407 sequence encoding permutein proteinSEQ ID NO: 259 Permutein protein encoded by pMON37407 sequence SEQ IDNO: 260 thermal amplification primer 60 SEQ ID NO: 261 thermalamplification primer 65 SEQ ID NO: 262 pMON37408 sequence encodingpermutein protein SEQ ID NO: 263 Permutein protein encoded by pMON37408sequence SEQ ID NO: 264 pMON40701 sequence encoding permutein proteinSEQ ID NO: 265 Permutein protein encoded by pMON40701 sequence SEQ IDNO: 266 thermal amplification primer Syn1 SEQ ID NO: 267 thermalamplification primer Syn2 SEQ ID NO: 268 thermal amplification primerSyn3 SEQ ID NO: 269 thermal amplification primer Syn4 SEQ ID NO: 270pMON40703 sequence encoding permutein protein SEQ ID NO: 271 Permuteinprotein encoded by pMON40703 sequence SEQ ID NO: 272 thermalamplification primer Syn10 SEQ ID NO: 273 thermal amplification primerSyn11 SEQ ID NO: 274 pMON40705 sequence encoding permutein protein SEQID NO: 275 Permutein protein encoded by pMON40705 sequence SEQ ID NO:276-277 Permutein linker sequences SEQ ID NO: 278 Patatin isozyme PatA+(including signal peptide) SEQ ID NO: 279 Patatin isozyme PatB+(including signal peptide) SEQ ID NO: 280 Patatin isozyme PatFm (matureprotein lacking signal peptide) SEQ ID NO: 281 Patatin isozyme PatIm(mature protein lacking signal peptide) SEQ ID NO: 282 Patatin isozymePatL+ (including signal peptide) SEQ ID NO: 283 Rational substitutionpeptide SEQ ID NO: 284 Corn homolog peptide SEQ ID NO: 285 patatinhomolog Pat17 DNA coding sequence and amino acid translation SEQ ID NO:286 patatin homolog Pat17 amino acid sequence SEQ ID NO: 287 dicotpatatin homolog amino acid sequence pentin1_phb SEQ ID NO: 288 dicotpatatin homolog amino acid sequence 5c9_phb SEQ ID NO: 289 maize patatinhomolog amino acid sequence corn1_pep SEQ ID NO: 290 maize patatinhomolog amino acid sequence corn2_pep SEQ ID NO: 291 maize patatinhomolog amino acid sequence corn3_pep SEQ ID NO: 292 maize patatinhomolog amino acid sequence corn4_pep SEQ ID NO: 293 maize patatinhomolog amino acid sequence corn5_pep

Definitions

[0032] The following definitions are provided in order to aid thoseskilled in the art in understanding the detailed description of thepresent invention. Some words and phrases may also be defined in othersections of the specification. No limitation should be placed on thedefinitions presented for the terms below, where other meanings areevidenced elseqhere in the specification in addition to those specifiedbelow.

[0033] “Allergen” refers to a biological or chemical substance thatinduces an allergic reaction or response. An allergic response can be animmunoglobulin E-mediated response.

[0034] Amino acid codes: A (Ala)=alanine; C (Cys)=cysteine; D(Asp)=aspartic acid; E (Glu)=glutamic acid; F (Phe)=phenylalanine; G(Gly)=glycine; H (His)=histidine; I (Ile)=isoleucine; K (Lys)=lysine; L(Leu)=leucine; M (Met)=methionine; N (Asn)=asparagine; P (Pro)=proline;Q (Gln)=glutamine; R (Arg)=arginine; S (Ser)=serine; T (Thr)=threonine;V=(Val) valine; W (Trp)=tryptophan; Y (Tyr)=tyrosine.

[0035] “Amplification: refers to increasing the number of copies of adesired molecule.

[0036] “Coding sequence”, “open reading frame”, and “structuralsequence” refer to the region of continuous sequential nucleic acid basepair triplets encoding a protein, polypeptide, or peptide sequence.

[0037] “Codon” refers to a sequence of three nucleotides that specify aparticular amino acid.

[0038] “Complementarity” refers to the specific binding of adenine tothymine (or uracil in RNA) and cytosine to guanine on opposite strandsof DNA or RNA.

[0039] “Deallergenize” (render hypoallergenic) refers to the method ofengineering or modifying a protein or the encoding DNA such that theprotein has a reduced or eliminated ability to induce an allergicresponse with respect to the ability of the unmodified protein. Adeallergenized protein can be referred to as being hypoallergenic. Thedegree of deallergenization of a protein can be measured in vitro by thereduced binding of IgE antibodies.

[0040] “DNA segment heterologous to the promoter region” means that thecoding DNA segment does not exist in nature in the same gene with thepromoter to which it is now attached.

[0041] “DNA segment” refers to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species.

[0042] “Electroporation” refers to a method of introducing foreign DNAinto cells that uses a brief, high voltage DC (direct current) charge topermeabilize the host cells, causing them to take up extra-chromosomalDNA.

[0043] “Encoding DNA” refers to chromosomal DNA, plasmid DNA, cDNA, orsynthetic DNA which encodes any of the enzymes discussed herein.

[0044] “Endogenous” refers to materials originating from within anorganism or cell.

[0045] “Endonuclease” refers to an enzyme that hydrolyzes doublestranded DNA at internal locations.

[0046] “Epitope” refers to a region on an allergen that interacts withthe cells of the immune system. Epitopes are often further defined bythe type of antibody or cell with which they interact, e.g. if theregion reacts with B-cells or antibodies (IgE), it is called a B-cellepitope.

[0047] “Exogenous” refers to materials originating from outside of anorganism or cell. This typically applies to nucleic acid molecules usedin producing transformed or transgenic host cells and plants.

[0048] “Expressibly coupled” and “expressibly linked” refer to apromoter or promoter region and a coding or structural sequence in suchan orientation and distance that transcription of the coding orstructural sequence can be directed by the promoter or promoter region.

[0049] “Expression” refers to the transcription of a gene to produce thecorresponding mRNA and translation of this mRNA to produce thecorresponding gene product, i.e., a peptide, polypeptide, or protein.

[0050] “Heterologous DNA” refers to DNA from a source different thanthat of the recipient cell.

[0051] “Homologous DNA” refers to DNA from the same source as that ofthe recipient cell.

[0052] “Identity” refers to the degree of similarity between two nucleicacid or protein sequences. An alignment of the two sequences isperformed by a suitable computer program. A widely used and acceptedcomputer program for performing sequence alignments is CLUSTALW v1.6(Thompson, et al. Nucl. Acids Res., 22: 4673-4680, 1994). The number ofmatching bases or amino acids is divided by the total number of bases oramino acids, and multiplied by 100 to obtain a percent identity. Forexample, if two 580 base pair sequences had 145 matched bases, theywould be 25 percent identical. If the two compared sequences are ofdifferent lengths, the number of matches is divided by the shorter ofthe two lengths. For example, if there were 100 matched amino acidsbetween 200 and a 400 amino acid proteins, they are 50 percent identicalwith respect to the shorter sequence. If the shorter sequence is lessthan 150 bases or 50 amino acids in length, the number of matches aredivided by 150 (for nucleic acid bases) or 50 (for amino acids), andmultiplied by 100 to obtain a percent identity.

[0053] “IgE” (Immunoglobulin E) refers to a specific class ofimmunoglobulin secreted by B cells. IgE binds to specific receptors onMast cells. Interaction of an allergen with mast cell-bound IgE maytrigger allergic symptoms.

[0054] “Immunotherapy” refers to any type of treatment that targets theimmune system. Allergy immunotherapy is a treatment in which aprogressively increasing dose of an allergen is given in order to inducean immune response characterized by tolerance to the antigen/allergen,also known as desensitization.

[0055] “In vitro” refers to “in the laboratory” and/or “outside of aliving organism”.

[0056] “In vivo” refers to “in a living organism”.

[0057] “Insecticidal polypeptide” refers to a polypeptide havinginsecticidal properties that adversely affects the growth anddevelopment of insect pests.

[0058] “Monocot” refers to plants having a single cotyledon (the firstleaf of the embryo of seed plants); examples include cereals such asmaize, rice, wheat, oats, and barley.

[0059] “Multiple cloning site” refers to an artificially constructedcollection of restriction enzyme sites in a vector that facilitatesinsertion of foreign DNA into the vector.

[0060] “Mutation” refers to any change or alteration in a nucleic acidsequence. Several types exist, including point, frame shift, splicing,and insertion/deletions.

[0061] “Native” refers to “naturally occurring in the same organism”.For example, a native promoter is the promoter naturally foundoperatively linked to a given coding sequence in an organism. A nativeprotein is one naturally found in nature and untouched or not otherwisemanipulated by the hand of man.

[0062] “Nucleic acid segment” is a nucleic acid molecule that has beenisolated free of total genomic DNA of a particular species, or that hasbeen synthesized. Included with the term “nucleic acid segment” are DNAsegments, recombinant vectors, plasmids, cosmids, phagemids, phage,viruses, etcetera.

[0063] “Nucleic acid” refers to deoxyribonucleic acid (DNA) andribonucleic acid (RNA).

[0064] Nucleic acid codes: A=adenosine; C=cytosine; G=guanosine;T=thymidine; N=equimolar A, C, G, and T; I=deoxyinosine; K=equimolar Gand T; R=equimolar A and G; S=equimolar C and G; W=equimolar A and T;Y=equimolar C and T.

[0065] “Open reading frame (ORF)” refers to a region of DNA or RNAencoding a peptide, polypeptide, or protein or capable of beingtranslated to protein, or a regioiu of DNA capable of being transcribedinto an RNA product.

[0066] “Plasmid” refers to a circular, extrachromosomal,self-replicating piece of DNA.

[0067] “Point mutation” refers to an alteration of a single nucleotidein a nucleic acid sequence.

[0068] “Polymerase chain reaction (PCR)” refers to an enzymatictechnique to create multiple copies of one sequence of nucleic acid.Copies of DNA sequence are prepared by shuttling a DNA polymerasebetween two oligonucleotides. The basis of this amplification method ismultiple cycles of temperature changes to denature, then re-annealamplimers, followed by extension to synthesize new DNA strands in theregion located between the flanking amplimers. Also known as thermalamplification.

[0069] “Probe” refers to a polynucleotide sequence which iscomplementary to a target polynucleotide sequence in the analyte. Anantibody can also be used as a probe to detect the presence of anantigen. In that sense, the antigen binding domain of the antibody hassome detectable affinity for the antigen and binds thereto. The bindingof the antibody to the antigen can be measured by means known in theart, such as by chemiluminescence, phosphorescence, flourescence,colorimetric chemical deposition at the site of binding, or otherwise.

[0070] “Promoter” or “promoter region” refers to a DNA sequence, usuallyfound upstream (5′) to a coding sequence, that controls expression ofthe coding sequence by controlling production of messenger RNA (mRNA) byproviding the recognition site for RNA polymerase and/or other factorsnecessary for start of transcription at the correct site. Ascontemplated herein, a promoter or promoter region includes variationsof promoters derived by means of ligation to various regulatorysequences, random or controlled mutagenesis, and addition or duplicationof enhancer sequences. The promoter region disclosed herein, andbiologically functional equivalents thereof, are responsible for drivingthe transcription of coding sequences under their control whenintroduced into a host as part of a suitable recombinant vector, asdemonstrated by its ability to produce mRNA.

[0071] “Recombinant DNA construct” or “recombinant vector” refers to anyagent such as a plasmid, cosmid, virus, autonomously replicatingsequence, phage, or linear or circular single-stranded ordouble-stranded DNA or RNA nucleotide sequence, derived from any source,capable of genomic integration or autonomous replication, comprising aDNA molecule in which one or more DNA sequences have been linked in afunctionally operative manner. Such recombinant DNA constructs orvectors are capable of introducing a 5′ regulatory sequence or promoterregion and a DNA sequence for a selected gene product into a cell insuch a manner that the DNA sequence is transcribed into a functionalmRNA which is translated and therefore expressed. Recombinant DNAconstructs or recombinant vectors can be constructed to be capable ofexpressing antisense RNAs, in order to inhibit translation of a specificRNA of interest.

[0072] “Recombinant proteins”, also referred to as “heterologousproteins”, are proteins which are normally not produced by the hostcell.

[0073] “Regeneration” refers to the process of growing a plant from aplant cell (e.g., plant protoplast or explant).

[0074] “Regeneration” refers to the process of growing a plant from aplant cell (e.g., plant protoplast or explant).

[0075] “Regulatory sequence” refers to a nucleotide sequence locatedupstream (5′), within, and/or downstream (3′) to a DNA sequence encodinga selected gene product whose transcription and expression is controlledby the regulatory sequence in conjunction with the protein syntheticapparatus of the cell.

[0076] “Restriction enzyme” refers to an enzyme that recognizes aspecific palindromic sequence of nucleotides in double stranded DNA andcleaves both strands; also called a restriction endonuclease. Cleavagetypically occurs within the restriction site.

[0077] “Result-effective substitution” (RES) refers to an amino acidsubstitution within an IgE-binding region (epitope) of a target proteinwhich reduces or eliminates the IgE binding by that epitope. Examplesherein are directed to patatin protein and homologues, however, as willbe readily recognized by those skilled in the art, the method is morebroadly applicable to proteins other than patatins, and in particular isapplicable to any protein exhibiting allergen eliciting properties.

[0078] “Selectable marker” refers to a nucleic acid sequence whoseexpression confers a phenotype facilitating identification of cellscontaining the nucleic acid sequence. Selectable markers include thosewhich confer resistance to toxic chemicals (e.g. ampicillin resistance,kanamycin resistance), complement a nutritional deficiency (e.g. uracil,histidine, leucine), or impart a visually distinguishing characteristic(e.g. color changes or fluorescence).

[0079] “Transcription” refers to the process of producing an RNA copyfrom a DNA template.

[0080] “Transformation” refers to a process of introducing an exogenousnucleic acid sequence (e.g., a vector, recombinant nucleic acidmolecule) into a cell or protoplast in which that exogenous nucleic acidis incorporated into a chromosome or is capable of autonomousreplication.

[0081] “Transformed cell” is a cell whose DNA has been altered by theintroduction of an exogenous nucleic acid molecule into that cell.

[0082] “Transgenic cell” refers to any cell derived from or regeneratedfrom a transformed cell or derived from a transgenic cell. Exemplarytransgenic cells include plant calli derived from a transformed plantcell and particular cells such as leaf, root, stem, e.g., somatic cells,or reproductive (germ) cells obtained from a transgenic plant.

[0083] “Transgenic plant” refers to a plant or progeny thereof derivedfrom a transformed plant cell or protoplast, wherein the plant DNAcontains an introduced exogenous nucleic acid sequence not originallypresent in a native, non-transgenic plant of the same species.Alternatively, the plant DNA can contain the introduced nucleic acidsequence in a higher copy number than in the native, non-transgenicplant of the same species.

[0084] “Translation” refers to the production of protein from messengerRNA.

[0085] “Vector” refers to a plasmid, cosmid, bacteriophage, or virusthat carries foreign DNA into a host organism.

[0086] “Western blot” refers to protein or proteins that have beenseparated by electrophoresis, transferred and immobilized onto a solidsupport, then probed with an antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0087] Design of Deallergenized Patatin Proteins

[0088] Deallergenizing a protein can be accomplished by theidentification of allergenic sites, followed by modification of thesites to reduce or eliminate the binding of antibodies to the sites. TheIgE-binding regions of patatin were previously unreported. Mapping ofthe IgE epitopes was accomplished by synthesizing 10-mer peptides basedon the patatin 17 protein sequence (SEQ ID NO: 2) which overlap by sixamino acids. As potato proteins are denatured upon cooking potatoproducts, it is expected that the 10-mer peptides sufficiently mimic theunfolded full length protein for antibody binding purposes. Peptideswere identified based upon their ability to bind to IgE antibodies.Individual amino acids within the identified peptides were changed toreduce or eliminate binding to IgE present in sera from potato sensitivepatients. These changes are termed result-effective amino acidsubstitutions (RES). The RES can be subsequently introduced into thefull length protein by site directed mutagenesis of the encoding nucleicacid sequence or other means known in the art. Similar strategies havebeen employed elsewhere to determine the dominant IgE epitopes in amajor peanut allergen (Stanley, J. S., et al., Arch. Biochem. Biophys.,342(2): 244-253, 1997).

[0089] Certain amino acid residues important for allergenicity ofpatatin are identified. Some of the designed patatin peptides whereinsingle amino acid residues were replaced with alanine or phenylalanine,showed significantly reduced or no binding to sera from potato sensitivepatients.

[0090] A “deallergenized patatin” refers to a patatin protein differingin at least one of the amino acid residues as defined by the resulteffective substitutions resulting in the patatin protein having reducedreactivity towards sera from potato sensitive patients. Thedeallergenized patatin preferably maintains insecticidal properties, andpreferably maintains its characteristic enzymatic profile.

[0091] Summary of Method to Deallergenize a Patatin Protein

[0092] Mapping of IgE epitopes by immunoassay of synthetic overlappingpeptides using sera from potato sensitive patients;

[0093] Identification of result-effective substitutions by alaninescanning and/or rational scanning;

[0094] Modification of the amino acid sequence of patatin bysite-directed mutagenesis of the encoding nucleic acid sequence;

[0095] Evaluation of enzyme activity (esterase) and/or insecticidalactivity of the modified protein(s); and

[0096] Evaluation of the new protein(s) for allergenicity by IgEimmunoassay.

[0097] Nucleic acid sequences encoding patatin have been cloned byseveral investigators (e.g. Mignery, et al., Nucleic Acids Research, 12:7987-8000, 1984; Mignery, et al., Gene, 62: 27-44, 1988; WO 94/21805;Canadian Patent Application No. 2090552). These nucleic acid sequencescan then be manipulated using site directed mutagenesis to encode ahypoallergenic patatin. These nucleic acid sequences can than be used totransform bacterial, yeast or plant cells, resulting in the productionof hypoallergenic patatin protein.

[0098] Deallergenized Patatin Proteins

[0099] For simplicity, individual amino acids are referred to by theirsingle letter codes. Correlation between the single letter codes, threeletter codes, and full amino acid names is presented in the definitionssection above.

[0100] One embodiment of the invention is an isolated deallergenizedpatatin protein. The protein is modified relative to the wild-typeprotein sequence such that they exhibit reduced binding to anti-patatinantibodies such as those obtained from humans or animals allergic topotatoes. The reduced binding is measured relative to the binding of theunmodified patatin protein to the anti-patatin antibodies.

[0101] The deallergenized patatin protein can comprise SEQ ID NO:2modified in one or more of the following regions, or SEQ ID NO:7modified in one or more of the following regions. The single or multipleamino acid modifications reduce the binding of the modified proteinrelative to the binding of the corresponding unmodified protein. Theregions for modification include amino acid positions 104-113 of SEQ IDNO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ IDNO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). Thepossible amino acid modifications include replacing an amino acid withA, E, F, P, or S. The modifications replace one or more amino acids inthe identified regions, without increasing or decreasing the totalnumber of amino acids in the protein.

[0102] Preferably, the deallergenized patatin protein comprises SEQ IDNO:2 modified by one or more changes, or SEQ ID NO:7 modified by one ormore changes. SEQ ID NO:7 differs from wild type SEQ ID NO:2 in that thefirst 22 amino acids of SEQ ID NO:2 are replaced with EAE (Glu-Ala-Glu).For example, the changes to SEQ ID NO:2 or SEQ ID NO:7 can be: the Ycorresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ IDNO:7 is replaced with F or A; the I corresponding to position 113 of SEQID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 137 of SEQID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the Scorresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 185 of SEQ IDNO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the Acorresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ IDNO:7 is replaced with S; the T corresponding to position 192 of SEQ IDNO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.

[0103] More preferably, SEQ ID NO:2 is modified by the following changesor SEQ ID NO:7 is modified by the following changes: the Y correspondingto position 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 193 of SEQ ID NO:2 or position 174 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 270 ofSEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

[0104] Most preferably, SEQ ID NO:2 is modified by the following changesor SEQ ID NO:7 is modified by the following changes: the Y correspondingto position 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 isreplaced with F; the Y corresponding to position 193 of SEQ ID NO:2 orposition 174 of SEQ ID NO:7 is replaced with F; the Y corresponding toposition 270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 316 of SEQ ID NO:2 or position297 of SEQ ID NO:7 is replaced with F; and the Y corresponding toposition 362 of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replacedwith F.

[0105] Nucleic Acids

[0106] An additional embodiment of the invention is an isolated nucleicacid molecule segment comprising a structural nucleic acid sequencewhich encodes a deallergenized patatin protein.

[0107] The structural nucleic acid sequence can generally encode anydeallergenized patatin protein. The structural nucleic acid sequencepreferably encodes a deallergenized patatin protein comprising SEQ IDNO:2 modified in one or more of the following regions, or SEQ ID NO:7modified in one or more of the following regions. The single or multipleamino acid modifications reduce the binding of the modified proteinrelative to the binding of the corresponding unmodified protein. Theregions for modification include amino acid positions 104-113 of SEQ IDNO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ IDNO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). Thepossible amino acid modifications include replacing an amino acid withA, E, F, P, or S. The modifications replace one or more amino acids inthe identified regions, without increasing or decreasing the totalnumber of amino acids in the protein.

[0108] Alternatively, the structural nucleic acid sequence encodes SEQID NO:2 modified by one or more of the following changes or encoding SEQID NO:7 modified by one or more of the following changes: the Ycorresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ IDNO:7 is replaced with F or A; the I corresponding to position 113 of SEQID NO:2 or position 94 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 129 of SEQ ID NO:2 or position 110 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 137 of SEQID NO:2 or position 118 of SEQ ID NO:7 is replaced with A; the Scorresponding to position 184 of SEQ ID NO:2 or position 165 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 185 of SEQ IDNO:2 or position 166 of SEQ ID NO:7 is replaced with F or A; the Acorresponding to position 188 of SEQ ID NO:2 or position 169 of SEQ IDNO:7 is replaced with S; the T corresponding to position 192 of SEQ IDNO:2 or position 173 of SEQ ID NO:7 is replaced with A or P; the Ycorresponding to position 193 of SEQ ID NO:2 or position 174 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 268 of SEQID NO:2 or position 249 of SEQ ID NO:7 is replaced with A or E; the Tcorresponding to position 269 of SEQ ID NO:2 or position 250 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 270 of SEQ IDNO:2 or position 251 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 273 of SEQ ID NO:2 or position 254 of SEQ IDNO:7 is replaced with A; the K corresponding to position 313 of SEQ IDNO:2 or position 294 of SEQ ID NO:7 is replaced with E; the Ncorresponding to position 314 of SEQ ID NO:2 or position 295 of SEQ IDNO:7 is replaced with A; the N corresponding to position 315 of SEQ IDNO:2 or position 296 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F or A; the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F; the Kcorresponding to position 367 of SEQ ID NO:2 or position 348 of SEQ IDNO:7 is replaced with A; the R corresponding to position 368 of SEQ IDNO:2 or position 349 of SEQ ID NO:7 is replaced with A; the Fcorresponding to position 369 of SEQ ID NO:2 or position 350 of SEQ IDNO:7 is replaced with A; the K corresponding to position 371 of SEQ IDNO:2 or position 352 of SEQ ID NO:7 is replaced with A; the Lcorresponding to position 372 of SEQ ID NO:2 or position 353 of SEQ IDNO:7 is replaced with A; and the L corresponding to position 373 of SEQID NO:2 or position 354 of SEQ ID NO:7 is replaced with A.

[0109] More preferably, the structural nucleic acid sequence encodes SEQID NO:2 modified by the following changes or SEQ ID NO:7 modified by thefollowing changes: the Y corresponding to position 106 of SEQ ID NO:2 orposition 87 of SEQ ID NO:7 is replaced with F; the Y corresponding toposition 129 of SEQ ID NO:2 or position 110 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 185 of SEQ ID NO:2 or position166 of SEQ ID NO:7 is replaced with F; the Y corresponding to position193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 270 of SEQ ID NO:2 or position 251 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 316 ofSEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

[0110] Most preferably, the structural nucleic acid sequence encodes SEQID NO:2 modified by the following changes or SEQ ID NO:7 modified by thefollowing changes: the Y corresponding to position 185 of SEQ ID NO:2 orposition 166 of SEQ ID NO:7 is replaced with F; the Y corresponding toposition 193 of SEQ ID NO:2 or position 174 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 270 of SEQ ID NO:2 or position251 of SEQ ID NO:7 is replaced with F; the Y corresponding to position316 of SEQ ID NO:2 or position 297 of SEQ ID NO:7 is replaced with F;and the Y corresponding to position 362 of SEQ ID NO:2 or position 343of SEQ ID NO:7 is replaced with F.

[0111] Recombinant Vectors

[0112] An additional embodiment is directed towards recombinant vectorscomprising a structural nucleic acid sequence which encodes adeallergenized patatin protein. The recombinant vector comprisesoperatively linked in the 5′ to 3′ orientation: a promoter that directstranscription of a structural nucleic acid sequence; a structuralnucleic acid sequence, and a 3′ transcription terminator.

[0113] The structural nucleic acid sequence can encode SEQ ID NO:2modified in one or more of the following regions, or SEQ ID NO:7modified in one or more of the following regions. The single or multipleamino acid modifications reduce the binding of the modified proteinrelative to the binding of the corresponding unmodified protein. Theregions for modification include amino acid positions 104-113 of SEQ IDNO:2 (85-94 of SEQ ID NO:7), 128-137 of SEQ ID NO:2 (109-118 of SEQ IDNO:7), 184-197 of SEQ ID NO:2 (165-178 of SEQ ID NO:7), 264-277 of SEQID NO:2 (245-258 of SEQ ID NO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQID NO:7), and 360-377 of SEQ ID NO:2 (341-358 of SEQ ID NO:7). Thepossible amino acid modifications include replacing an amino acid withA, E, F, P, or S. The modifications replace one or more amino acids inthe identified regions, without increasing or decreasing the totalnumber of amino acids in the protein.

[0114] Alternatively, the recombinant vector comprises operativelylinked in the 5′ to 3′ orientation: a promoter that directstranscription of a structural nucleic acid sequence; a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by one or more ofthe following changes or encoding SEQ ID NO:7 modified by one or more ofthe following changes: the Y corresponding to position 106 of SEQ IDNO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the Icorresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ IDNO:7 is replaced with A; the S corresponding to position 184 of SEQ IDNO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F or A; the A corresponding to position 188 of SEQID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the Tcorresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ IDNO:7 is replaced with A or P; the Y corresponding to position 193 of SEQID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ IDNO:7 is replaced with A or E; the T corresponding to position 269 of SEQID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 273 of SEQID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ IDNO:7 is replaced with E; the N corresponding to position 314 of SEQ IDNO:2 or position 295 of SEQ ID NO:7 is replaced with A; the Ncorresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F; the K corresponding to position 367 of SEQ IDNO:2 or position 348 of SEQ ID NO:7 is replaced with A; the Rcorresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ IDNO:7 is replaced with A; the F corresponding to position 369 of SEQ IDNO:2 or position 350 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ IDNO:7 is replaced with A; the L corresponding to position 372 of SEQ IDNO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the Lcorresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ IDNO:7 is replaced with A; and a 3′ transcription terminator.

[0115] More preferably, the vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified by the following changes or SEQID NO:7 modified by the following changes: the Y corresponding toposition 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 193 of SEQ ID NO:2 or position 174 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 270 ofSEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

[0116] Most preferably, the vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified by the following changes or SEQID NO:7 modified by the following changes: the Y corresponding toposition 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 193 of SEQ ID NO:2 or position174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 316 of SEQ ID NO:2 or position 297 ofSEQ ID NO:7 is replaced with F; and the Y corresponding to position 362of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

[0117] Recombinant Host Cells

[0118] A further embodiment of the invention is directed towardsrecombinant host cells comprising a structural nucleic acid sequenceencoding a deallergenized patatin protein. The recombinant host cellpreferably produces a deallergenized patatin protein. More preferably,the recombinant host cell produces a deallergenized patatin protein in aconcentration sufficient to inhibit growth or to kill an insect whichingests the recombinant host cell. The recombinant host cell cangenerally comprise any structural nucleic acid sequence encoding adeallergenized patatin protein.

[0119] The recombinant host cell can comprise a structural nucleic acidsequence encoding SEQ ID NO:2 modified in one or more of the followingregions, or SEQ ID NO:7 modified in one or more of the followingregions. The single or multiple amino acid modifications reduce thebinding of the modified protein relative to the binding of thecorresponding unmodified protein. The regions for modification includeamino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7),128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2(165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ IDNO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 ofSEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acidmodifications include replacing an amino acid with A, E, F, P, or S. Themodifications replace one or more amino acids in the identified regions,without increasing or decreasing the total number of amino acids in theprotein.

[0120] Alternatively, the recombinant host cell comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by one or more ofthe following changes or encoding SEQ ID NO:7 modified by one or more ofthe following changes: the Y corresponding to position 106 of SEQ IDNO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the Icorresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ IDNO:7 is replaced with A; the S corresponding to position 184 of SEQ IDNO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F or A; the A corresponding to position 188 of SEQID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the Tcorresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ IDNO:7 is replaced with A or P; the Y corresponding to position 193 of SEQID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ IDNO:7 is replaced with A or E; the T corresponding to position 269 of SEQID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 273 of SEQID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ IDNO:7 is replaced with E; the N corresponding to position 314 of SEQ IDNO:2 or position 295 of SEQ ID NO:7 is replaced with A; the Ncorresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F; the K corresponding to position 367 of SEQ IDNO:2 or position 348 of SEQ ID NO:7 is replaced with A; the Rcorresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ IDNO:7 is replaced with A; the F corresponding to position 369 of SEQ IDNO:2 or position 350 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ IDNO:7 is replaced with A; the L corresponding to position 372 of SEQ IDNO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the Lcorresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ IDNO:7 is replaced with A.

[0121] More preferably, the recombinant host cell comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by the followingchanges or SEQ ID NO:7 modified by the following changes: the Ycorresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 193 of SEQ IDNO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

[0122] Most preferably, the recombinant host cell comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by the followingchanges or SEQ ID NO:7 modified by the following changes: the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 193 of SEQ IDNO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

[0123] The recombinant host cell can generally be any type of host cell,and preferably is a bacterial, fungal, or plant cell. The bacterial cellis preferably an Escherichia coli bacterial cell. The fungal cell ispreferably a Saccharomyces cerevisiae, Schizosaccharomyces pombe, orPichia pastoris fungal cell. The plant cell can be a monocot, dicot, orconifer plant cell. The plant cell is preferably an alfalfa, banana,canola, corn, cotton, cucumber, peanut, potato, rice, soybean,sunflower, sweet potato, tobacco, tomato, or wheat plant cell. Therecombinant host cell preferably further comprises operatively linked tothe structural nucleic acid sequence a promoter that directstranscription of the structural nucleic acid sequence. The recombinanthost cell preferably further comprises operatively linked to thestructural nucleic acid sequence a 3′ transcription terminator and apolyadenylation site.

[0124] Recombinant Plants

[0125] An additional embodiment of the invention is a recombinant plantcomprising a structural nucleic acid sequence encoding a deallergenizedpatatin protein. The recombinant plant preferably produces adeallergenized patatin protein. More preferably, the recombinant plantproduces a deallergenized patatin protein in a concentration sufficientto inhibit growth or to kill an insect which ingests plant tissue fromthe recombinant plant.

[0126] The recombinant plant can comprise a structural nucleic acidsequence encoding SEQ ID NO:2 modified in one or more of the followingregions, or SEQ ID NO:7 modified in one or more of the followingregions. The single or multiple amino acid modifications reduce thebinding of the modified protein relative to the binding of thecorresponding unmodified protein. The regions for modification includeamino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7),128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2(165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ IDNO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 ofSEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acidmodifications include replacing an amino acid with A, E, F, P, or S. Themodifications replace one or more amino acids in the identified regions,without increasing or decreasing the total number of amino acids in theprotein.

[0127] Alternatively, the recombinant plant can comprise a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by one or more ofthe following changes or encoding SEQ ID NO:7 modified by one or more ofthe following changes: the Y corresponding to position 106 of SEQ IDNO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the Icorresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ IDNO:7 is replaced with A; the S corresponding to position 184 of SEQ IDNO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F or A; the A corresponding to position 188 of SEQID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the Tcorresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ IDNO:7 is replaced with A or P; the Y corresponding to position 193 of SEQID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ IDNO:7 is replaced with A or E; the T corresponding to position 269 of SEQID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 273 of SEQID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ IDNO:7 is replaced with E; the N corresponding to position 314 of SEQ IDNO:2 or position 295 of SEQ ID NO:7 is replaced with A; the Ncorresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F; the K corresponding to position 367 of SEQ IDNO:2 or position 348 of SEQ ID NO:7 is replaced with A; the Rcorresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ IDNO:7 is replaced with A; the F corresponding to position 369 of SEQ IDNO:2 or position 350 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ IDNO:7 is replaced with A; the L corresponding to position 372 of SEQ IDNO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the Lcorresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ IDNO:7 is replaced with A.

[0128] More preferably, the recombinant plant comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by the followingchanges or SEQ ID NO:7 modified by the following changes: the Ycorresponding to position 106 of SEQ ID NO:2 or position 87 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 193 of SEQ IDNO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

[0129] Most preferably, the recombinant plant comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by the followingchanges or SEQ ID NO:7 modified by the following changes: the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 193 of SEQ IDNO:2 or position 174 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F; and the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F.

[0130] The recombinant plant can generally be any type of plant. Theplant can be a monocot, dicot, or conifer plant. The plant is preferablyan alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato,rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.

[0131] The recombinant plant preferably further comprises operativelylinked to the structural nucleic acid sequence a promoter that directstranscription of the structural nucleic acid sequence. The recombinantplant preferably further comprises operatively linked to the structuralnucleic acid sequence a 3′ transcription terminator and apolyadenylation site.

[0132] Methods of Preparation

[0133] Embodiments of the invention are further directed towards methodsof preparing recombinant host cells and recombinant plants useful forthe production of deallergenized patatin proteins.

[0134] A method of preparing a recombinant host cell useful for theproduction of deallergenized patatin proteins can comprise selecting ahost cell; transforming the host cell with a recombinant vector; andobtaining recombinant host cells.

[0135] The recombinant vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified in one or more of the followingregions, or SEQ ID NO:7 modified in one or more of the followingregions. The single or multiple amino acid modifications reduce thebinding of the modified protein relative to the binding of thecorresponding unmodified protein. The regions for modification includeamino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7),128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2(165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ IDNO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 ofSEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acidmodifications include replacing an amino acid with A, E, F, P, or S. Themodifications replace one or more amino acids in the identified regions,without increasing or decreasing the total number of amino acids in theprotein.

[0136] Alternatively, the recombinant vector comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by one or more ofthe following changes or encoding SEQ ID NO:7 modified by one or more ofthe following changes: the Y corresponding to position 106 of SEQ IDNO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the Icorresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ IDNO:7 is replaced with A; the S corresponding to position 184 of SEQ IDNO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F or A; the A corresponding to position 188 of SEQID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the Tcorresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ IDNO:7 is replaced with A or P; the Y corresponding to position 193 of SEQID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ IDNO:7 is replaced with A or E; the T corresponding to position 269 of SEQID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 273 of SEQID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ IDNO:7 is replaced with E; the N corresponding to position 314 of SEQ IDNO:2 or position 295 of SEQ ID NO:7 is replaced with A; the Ncorresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F; the K corresponding to position 367 of SEQ IDNO:2 or position 348 of SEQ ID NO:7 is replaced with A; the Rcorresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ IDNO:7 is replaced with A; the F corresponding to position 369 of SEQ IDNO:2 or position 350 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ IDNO:7 is replaced with A; the L corresponding to position 372 of SEQ IDNO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the Lcorresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ IDNO:7 is replaced with A.

[0137] More preferably, the vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified by the following changes or SEQID NO:7 modified by the following changes: the Y corresponding toposition 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 193 of SEQ ID NO:2 or position 174 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 270 ofSEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

[0138] Most preferably, the vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified by the following changes or SEQID NO:7 modified by the following changes: the Y corresponding toposition 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 193 of SEQ ID NO:2 or position174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 316 of SEQ ID NO:2 or position 297 ofSEQ ID NO:7 is replaced with F; and the Y corresponding to position 362of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

[0139] The method can generally be used to prepare any type ofrecombinant host cell. Preferably, the method can be used to prepare arecombinant bacterial cell, a recombinant fungal cell, or a recombinantplant cell. The bacterial cell is preferably an Escherichia colibacterial cell. The fungal cell is preferably a Saccharomycescerevisiae, Schizosaccharomyces pombe, or Pichia pastoris fungal cell.The plant cell can be a monocot, dicot, or conifer plant cell. The plantcell is preferably an alfalfa, banana, canola, corn, cotton, cucumber,peanut, potato, rice, soybean, sunflower, sweet potato, tobacco, tomato,or wheat plant cell.

[0140] An additional embodiment is directed towards methods for thepreparation of recombinant plants useful for the production ofdeallergenized patatin proteins. The method can comprise selecting ahost plant cell; transforming the host plant cell with a recombinantvector; obtaining recombinant host cells; and regenerating a recombinantplant from the recombinant host plant cells.

[0141] The recombinant vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified in one or more of the followingregions, or SEQ ID NO:7 modified in one or more of the followingregions. The single or multiple amino acid modifications reduce thebinding of the modified protein relative to the binding of thecorresponding unmodified protein. The regions for modification includeamino acid positions 104-113 of SEQ ID NO:2 (85-94 of SEQ ID NO:7),128-137 of SEQ ID NO:2 (109-118 of SEQ ID NO:7), 184-197 of SEQ ID NO:2(165-178 of SEQ ID NO:7), 264-277 of SEQ ID NO:2 (245-258 of SEQ IDNO:7), 316-325 of SEQ ID NO:2 (297-306 of SEQ ID NO:7), and 360-377 ofSEQ ID NO:2 (341-358 of SEQ ID NO:7). The possible amino acidmodifications include replacing an amino acid with A, E, F, P, or S. Themodifications replace one or more amino acids in the identified regions,without increasing or decreasing the total number of amino acids in theprotein.

[0142] Alternatively, the recombinant vector comprises a structuralnucleic acid sequence encoding SEQ ID NO:2 modified by one or more ofthe following changes or encoding SEQ ID NO:7 modified by one or more ofthe following changes: the Y corresponding to position 106 of SEQ IDNO:2 or position 87 of SEQ ID NO:7 is replaced with F or A; the Icorresponding to position 113 of SEQ ID NO:2 or position 94 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 129 of SEQ IDNO:2 or position 110 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 137 of SEQ ID NO:2 or position 118 of SEQ IDNO:7 is replaced with A; the S corresponding to position 184 of SEQ IDNO:2 or position 165 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 185 of SEQ ID NO:2 or position 166 of SEQ IDNO:7 is replaced with F or A; the A corresponding to position 188 of SEQID NO:2 or position 169 of SEQ ID NO:7 is replaced with S; the Tcorresponding to position 192 of SEQ ID NO:2 or position 173 of SEQ IDNO:7 is replaced with A or P; the Y corresponding to position 193 of SEQID NO:2 or position 174 of SEQ ID NO:7 is replaced with F or A; the Kcorresponding to position 268 of SEQ ID NO:2 or position 249 of SEQ IDNO:7 is replaced with A or E; the T corresponding to position 269 of SEQID NO:2 or position 250 of SEQ ID NO:7 is replaced with A; the Ycorresponding to position 270 of SEQ ID NO:2 or position 251 of SEQ IDNO:7 is replaced with F or A; the K corresponding to position 273 of SEQID NO:2 or position 254 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 313 of SEQ ID NO:2 or position 294 of SEQ IDNO:7 is replaced with E; the N corresponding to position 314 of SEQ IDNO:2 or position 295 of SEQ ID NO:7 is replaced with A; the Ncorresponding to position 315 of SEQ ID NO:2 or position 296 of SEQ IDNO:7 is replaced with A; the Y corresponding to position 316 of SEQ IDNO:2 or position 297 of SEQ ID NO:7 is replaced with F or A; the Ycorresponding to position 362 of SEQ ID NO:2 or position 343 of SEQ IDNO:7 is replaced with F; the K corresponding to position 367 of SEQ IDNO:2 or position 348 of SEQ ID NO:7 is replaced with A; the Rcorresponding to position 368 of SEQ ID NO:2 or position 349 of SEQ IDNO:7 is replaced with A; the F corresponding to position 369 of SEQ IDNO:2 or position 350 of SEQ ID NO:7 is replaced with A; the Kcorresponding to position 371 of SEQ ID NO:2 or position 352 of SEQ IDNO:7 is replaced with A; the L corresponding to position 372 of SEQ IDNO:2 or position 353 of SEQ ID NO:7 is replaced with A; and the Lcorresponding to position 373 of SEQ ID NO:2 or position 354 of SEQ IDNO:7 is replaced with A.

[0143] More preferably, the vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified by the following changes or SEQID NO:7 modified by the following changes: the Y corresponding toposition 106 of SEQ ID NO:2 or position 87 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 129 of SEQ ID NO:2 or position110 of SEQ ID NO:7 is replaced with F; the Y corresponding to position185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 193 of SEQ ID NO:2 or position 174 ofSEQ ID NO:7 is replaced with F; the Y corresponding to position 270 ofSEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F; the Ycorresponding to position 316 of SEQ ID NO:2 or position 297 of SEQ IDNO:7 is replaced with F; and the Y corresponding to position 362 of SEQID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

[0144] Most preferably, the vector comprises a structural nucleic acidsequence encoding SEQ ID NO:2 modified by the following changes or SEQID NO:7 modified by the following changes: the Y corresponding toposition 185 of SEQ ID NO:2 or position 166 of SEQ ID NO:7 is replacedwith F; the Y corresponding to position 193 of SEQ ID NO:2 or position174 of SEQ ID NO:7 is replaced with F; the Y corresponding to position270 of SEQ ID NO:2 or position 251 of SEQ ID NO:7 is replaced with F;the Y corresponding to position 316 of SEQ ID NO:2 or position 297 ofSEQ ID NO:7 is replaced with F; and the Y corresponding to position 362of SEQ ID NO:2 or position 343 of SEQ ID NO:7 is replaced with F.

[0145] The recombinant plant can generally be any type of plant. Theplant can be a monocot, dicot, or conifer plant. The plant is preferablyan alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato,rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.

[0146] Deallergenized patatin proteins can be prepared by isolating thedeallergenized patatin protein from any one of the above described hostcells or plants.

[0147] Deglycosylation

[0148] The examples herein provide evidence that glycosylation of cancontribute to the allergenicity of a protein. Accordingly, rationalsubstitution of amino acid residues likely to be the targets ofglycosylation within a subject allergen protein may reduce or eliminatethe allergenic properties of the protein without adversely affecting theenzymatic, insecticidal, antifungal or other functional properties ofthe protein.

[0149] Glycosylation commonly occurs as either N-linked or O-linkedforms. N-linked glycosylation usually occurs at the motifAsn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro (Kasturi, L. etal., Biochem J. 323: 415-519, 1997; Melquist, J. L. et al., Biochemistry37: 6833-6837, 1998). O-linked glycosylation occurs between the hydroxylgroup of serine or threonine and an amino sugar.

[0150] Site directed mutagenesis of selected asparagine, serine, orthreonine may be used to reduce or eliminate the glycosylation ofpatatin proteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motifreveals one occurrence at amino acid positions 202-204. Mutagenizationof the nucleic acid sequence encoding this region may result in areduced allergenicity of the encoded protein.

[0151] In order to test this conceptual approach to reducingallergenicity of patatin proteins, two sets of experiments wereperformed: a) production of patatin proteins in Escherichia coli, whichdo not glycosylate proteins; and b) production of patatin proteins withan N202Q site directed mutation.

[0152] Antibodies obtained from patients HS-07 and G15-MON (not potatoallergic) did not show specific binding to wild type patatin, patatinproduced in E. coli, or the N202Q variant. Antibodies obtained frompatient HS-01 (potato allergic) bound to wild type patatin, but not topatatin produced in E. coli or the N202Q variant. Antibodies obtainedfrom patient HS-02 (potato allergic) bound strongly to wild typepatatin, but extremely weakly to patatin produced in E. coli, andbinding to the N202Q variant resembled vector controls. Antibodiesobtained from patient HS-03 (potato allergic) bound to wild typepatatin, but not to patatin produced in E. coli or the N202Q variant.Antibodies obtained from patient HS-05 (potato allergic) bound to wildtype patatin, but very weakly to patatin produced in E. coli and theN202Q variant. Antibodies obtained from patient HS-06 (potato allergic)strongly bound wild type patatin, the N202Q variant, and to patatinproduced in E. coli. These results strongly suggest that glycosylationis at least partially responsible for the antigenic properties ofpatatin proteins, and that site directed mutagenesis may be used toreduce or eliminate specific antibody binding. Mutagenesis at position202 of SEQ ID NO:2 may be useful for reducing or eliminating specificantibody binding.

[0153] Permuteins

[0154] The positions of the internal breakpoints described in thefollowing Examples are found on the protein surface, and are distributedthroughout the linear sequence without any obvious bias towards the endsor the middle. Breakpoints occurring below the protein surface canadditionally be selected. The rearranged two subunits can be joined by apeptide linker. A preferred embodiment involves the linking of theN-terminal and C-terminal subunits by a three amino acid linker,although linkers of various sizes can be used. Additionally, theN-terminal and C-terminal subunits can be joined lacking a linkersequence. Furthermore, a portion of the C-terminal subunit can bedeleted and the connection made from the truncated C-terminal subunit tothe original N-terminal subunit and vice versa as previously described(Yang and Schachman, Proc. Natl. Acad. Sci. U.S.A., 90: 11980-11984,1993; Viguera, et al., Mol. Biol., 247: 670-681, 1995; Protasova, etal., Prot. Eng., 7: 1373-1377, 1994).

[0155] The novel insecticidal proteins of the present invention can berepresented by the formula:

X¹-(L)_(a)-X²

[0156] wherein;

[0157] a is 0 or 1, and if a is 0, then the permutein does not contain alinker sequence;

[0158] X¹ is a polypeptide sequence corresponding to amino acids n+1through J;

[0159] X² is a polypeptide corresponding to amino acids 1 through n;

[0160] n is an integer ranging from 1 to J-1;

[0161] J is an integer greater than n+1; and

[0162] L is a linker.

[0163] In the formula above, the constituent amino acid residues of thenovel insecticidal protein are numbered sequentially 1 through J fromthe original amino terminus to the original carboxyl terminus. A pair ofadjacent amino acids within this protein can be numbered n and n+1respectively where n is an integer ranging from 1 to J-1. The residuen+1 becomes the new N-terminus of the novel insecticidal protein and theresidue n becomes the new C-terminus of the novel insecticidal protein.

[0164] For example, a parent protein sequence consisting of 120 aminoacids can be selected as a starting point for designing a permutein(J=120). If the breakpoint is selected as being between position 40 andposition 41, then n=40. If a linker is selected to join the twosubunits, the resulting permutein will have the formula: (amino acids41-120)-L-(amino acids 1-40). If a linker was not used, the resultingpermutein will have the formula: (amino acids 41-120)-(amino acids1-40).

[0165] The length of the amino acid sequence of the linker can beselected empirically, by using structural information, or by using acombination of the two approaches. When no structural information isavailable, a small series of linkers can be made whose length can span arange of 0 to 50 Å and whose sequence is chosen in order to besubstantially consistent with surface exposure (Hopp and Woods, Mol.Immunol., 20: 483-489, 1983; Kyte and Doolittle, J. Mol. Biol., 157:105-132, 1982; Lee and Richards, J. Mol. Biol., 55: 379-400, 1971) andthe ability to adopt a conformation which does not significantly affectthe overall configuration of the protein (Karplus and Schulz,Naturwissenschaften, 72: 212-213, 1985). Assuming an average length of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween about 0 to about 30 residues, with 0 to about 15 residues beingthe preferred range. Accordingly, there are many such sequences thatvary in length or composition that can serve as linkers with the primaryconsideration being that they be neither excessively long norexcessively short (Sandhu, et al., Critical Rev. Biotech., 12: 437-467,1992). If the linker is too long, entropy effects may destabilize thethree-dimensional fold and may affect protein folding. If the linker istoo short, it may destabilize the molecule due to torsional or stericstrain.

[0166] Use of the distance between the chain ends, defined as thedistance between the C-alpha carbons, can be used to define the lengthof the sequence to be used, or at least to limit the number ofpossibilities that can be tested in an empirical selection of linkers.Using the calculated length as a guide, linkers with a range of numberof residues (calculated using 2 to 3.8 Å per residue) can be selected.These linkers can be composed of the original sequence, shortened orlengthened as necessary, and when lengthened the additional residues canbe chosen to be flexible and hydrophilic as described above; oroptionally the original sequence can be substituted for using a seriesof linkers, one example being Gly-Pro-Gly (SEQ ID NO:277); or optionallya combination of the original sequence and new sequence having theappropriate total length can be used. An alternative short, flexiblelinker sequence is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276).

[0167] Selection of Permutein Breakpoints

[0168] Sequences of novel patatin analogs capable of folding tobiologically active molecules can be prepared by appropriate selectionof the beginning (amino terminus) and ending (carboxyl terminus)positions from within the original polypeptide chain while optionallyusing a linker sequence as described above. Amino and carboxyl terminican be selected from within a common stretch of sequence, referred to asa breakpoint region, using the guidelines described below. A novel aminoacid sequence is thus generated by selecting amino and carboxyl terminifrom within the same breakpoint region. In many cases, the selection ofthe new termini will be such that the original position of the carboxylterminus immediately preceded that of the amino terminus. However,selections of termini anywhere within the region may result in afunctional protein, and that these will effectively lead to eitherdeletions or additions to the amino or carboxyl portions of the newsequence.

[0169] The primary amino acid sequence of a protein dictates folding tothe three-dimensional structure beneficial for expression of itsbiological function. It is possible to obtain and interpretthree-dimensional structural information using x-ray diffraction ofsingle protein crystals or nuclear magnetic resonance spectroscopy ofprotein solutions. Examples of structural information that are relevantto the identification of breakpoint regions include the location andtype of protein secondary structure (alpha and 3-10 helices, paralleland anti-parallel beta sheets, chain reversals and turns, and loops(Kabsch and Sander, Biopolymers, 22: 2577-2637, 1983), the degree ofsolvent exposure of amino acid residues, the extent and type ofinteractions of residues with one another (Chothia, C., Ann. Rev.Biochem., 53: 537-572, 1984), and the static and dynamic distribution ofconformations along the polypeptide chain (Alber and Mathews, MethodsEnzymol., 154: 511-533, 1987). In some cases additional information isknown about solvent exposure of residues, one example is a site ofpost-translational attachment of carbohydrate which is necessarily onthe surface of the protein. When experimental structural information isnot available, or when it is not feasible to obtain the information,methods are available to analyze the primary amino acid sequence inorder to make predictions of protein secondary and tertiary structure,solvent accessibility and the occurrence of turns and loops (Fasman, G.,Ed. Plenum, New York, 1989; Robson, B. and Gamier, J. Nature 361: 506,1993).

[0170] Biochemical methods can be applicable for empirically determiningsurface exposure when direct structural methods are not feasible; forexample, using the identification of sites of chain scission followinglimited proteolysis in order to infer surface exposure (Gentile, F. andSalvatore, G., Eur. J. Biochem., 218: 603-621, 1993). Thus, using eitherthe experimentally derived structural information or predictive methods(Srinivasan, R. and Rose, G. D. Proteins, 22: 81-99, 1995), the parentalamino acid sequence can be analyzed to classify regions according towhether or not they are integral to the maintenance of secondary andtertiary structure. The sequences within regions that are known to beinvolved in periodic secondary structure (alpha and 3-10 helices,parallel and anti-parallel beta sheets) are regions that should beavoided. Similarly, regions of amino acid sequence that are observed orpredicted to have a low degree of solvent exposure are more likely to bepart of the so-called hydrophobic core of the protein and should also beavoided for selection of amino and carboxyl termini. Regions that areknown or predicted to be in surface turns or loops, and especially thoseregions that are known not to be required for biological activity, canbe preferred sites for new amino and carboxyl termini. Stretches ofamino acid sequence that are preferred based on the above criteria canbe selected as breakpoint regions.

[0171] An embodiment of the invention is directed towards patatinpermutein proteins. The permutein proteins preferably maintain esteraseactivity and insecticidal properties. The permutein proteins preferablyare less allergenic than the wild type patatin protein to individuals oranimals allergic to potatoes. This can be assayed by the binding ofantibodies to the wild type patatin and patatin permutein proteins.

[0172] The permutein proteins can optionally contain a linker sequence.The linker can generally be any amino acid sequence, preferably isGly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) or Gly-Pro-Gly (SEQ IDNO:277), and more preferably is Gly-Pro-Gly (SEQ ID NO:277). Specificpermutein proteins comprise: (amino acids 247-386 of SEQ IDNO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids 269-386of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQ IDNO:247, and SEQ ID NO:259.

[0173] Embodiments of the invention also include isolated nucleic acidmolecule segments comprising a structural nucleic acid sequence encodinga patatin permutein protein. The encoded permutein protein can generallybe any permutein protein, and preferably comprises (amino acids 247-386of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2), (amino acids269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQ ID NO:2), SEQID NO:247, or SEQ ID NO:259. The linker can generally be any amino acidsequence, preferably is Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276) orGly-Pro-Gly (SEQ ID NO:277), and more preferably is Gly-Pro-Gly (SEQ IDNO:277). Alternatively, the encoded patatin permutein protein can lack alinker sequence. The structural nucleic acid sequence is preferably SEQID NO:246 or SEQ ID NO:258.

[0174] An embodiment of the invention is directed towards recombinantvectors which encode a patatin permutein protein. The vector cancomprise operatively linked in the 5′ to 3′ orientation: a promoter thatdirects transcription of a structural nucleic acid sequence; astructural nucleic acid sequence encoding a protein selected from thegroup consisting of: (amino acids 247-386 of SEQ ID NO:2)-linker-(aminoacids 24-246 of SEQ ID NO:2); and (amino acids 269-386 of SEQ IDNO:2)-linker-(amino acids 24-268 of SEQ ID NO:2); and a 3′ transcriptionterminator. The linker can comprise Gly-Pro-Gly (SEQ ID NO:277) orGly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively, the encodedpatatin permutein protein can lack a linker sequence. The structuralnucleic acid sequence can preferably be SEQ ID NO:246 or SEQ ID NO:258,and preferably encodes SEQ ID NO:247 or SEQ ID NO:259.

[0175] An additional embodiment of the invention is directed towardsrecombinant host cells useful for the production of a patatin permuteinprotein. The recombinant host cell preferably produces a patatinpermutein protein. More preferably, the recombinant host cell produces apatatin permutein protein in a concentration sufficient to inhibitgrowth or to kill an insect which ingests the recombinant host cell. Therecombinant host cell can comprise a structural nucleic acid sequenceencoding a protein selected from the group consisting of: (amino acids247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQ ID NO:2); and(amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids 24-268 of SEQID NO:2). The linker can generally be any amino acid sequence, andpreferably is Gly-Pro-Gly (SEQ ID NO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly(SEQ ID NO:276). Alternatively, the encoded patatin permutein proteincan lack a linker sequence. The structural nucleic acid sequence ispreferably SEQ ID NO:246 or SEQ ID NO:258, and preferably encodes SEQ IDNO:247 or SEQ ID NO:259. The structural nucleic acid sequence can beoperatively linked to a promoter sequence that directs transcription ofthe structural nucleic acid sequence, a 3′ transcription terminator, anda 3′ polyadenylation signal sequence. The recombinant host cell cangenerally be any type of host cell, and preferably is a bacterial,fungal, or plant host cell. The bacterial cell is preferably anEscherichia coli bacterial cell. The fungal cell is preferably aSaccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastorisfungal cell. The plant cell can be a monocot, dicot, or conifer plantcell. The plant cell is preferably an alfalfa, banana, canola, corn,cotton, cucumber, peanut, potato, rice, soybean, sunflower, sweetpotato, tobacco, tomato, or wheat plant cell.

[0176] An additional embodiment of the invention is directed towardsrecombinant plants which are useful for the production of patatinpermutein proteins. The recombinant plant preferably produces a patatinpermutein protein. More preferably, the recombinant plant produces apatatin permutein protein in a concentration sufficient to inhibitgrowth or to kill an insect which ingests tissue from the recombinantplant. The recombinant plant can comprise a structural nucleic acidsequence encoding a protein selected from the group consisting of:(amino acids 247-386 of SEQ ID NO:2)-linker-(amino acids 24-246 of SEQID NO:2); and (amino acids 269-386 of SEQ ID NO:2)-linker-(amino acids24-268 of SEQ ID NO:2). The linker can comprise Gly-Pro-Gly (SEQ IDNO:277) or Gly-Gly-Gly-Ser-Gly-Gly-Gly (SEQ ID NO:276). Alternatively,the encoded protein can lack a linker sequence. The structural nucleicacid sequence is preferably SEQ ID NO:246 or SEQ ID NO:258, andpreferably encodes SEQ ID NO:247 or SEQ ID NO:259. The structuralnucleic acid sequence can be operatively linked to a promoter sequencethat directs transcription of the structural nucleic acid sequence, a 3′transcription terminator, and a 3′ polyadenylation signal sequence. Therecombinant plant can generally be any type of plant, and preferably isan alfalfa, banana, canola, corn, cotton, cucumber, peanut, potato,rice, soybean, sunflower, sweet potato, tobacco, tomato, or wheat plant.

[0177] Permutein proteins can be prepared by isolating the permuteinprotein from any one of the above described host cells or plants.

[0178] Immunotherapy for Potato Allergy

[0179] Immunotherapy for food allergy has been largely unsuccessful dueto the lack of appropriate therapeutic reagents (Sampson, H. A., J.Allergy Clin. Immunol., 90(2): 151-152, 1992). Immunotherapy hastypically involved the administration (orally or by subcutaneousinjections) of increasing doses of crude protein extracts of theoffending allergenic entities which usually contain variable mixes ofmany different proteins (Scheiner, O., Wien Klin Wochenschr., 105(22):653-658, 1993). While there are reports of highly successful clinicalapplications of immunotherapy for food allergens (Romano, P. C., et al.,Allergol. Immunopathol. (Madr), 12(4): 275-281, 1984), those reports arerare and the clinical literature in general recommends avoidance farmore strongly than therapy (Gay, G., Allerg. Immunol. (Paris), 29(6):169-170, 1997). One of the primary reasons for the failure of manyclinical attempts to induce tolerance to allergens in general and foodallergens in particular relates to anecdotal comments by numerousallergists, that patients don't tolerate the doses of allergen requiredto achieve tolerance. Animal studies examining the relationship ofantigen dose and the induction of tolerance have demonstrated a strongpositive correlation (Chen, Y., et al., Proc. Natl. Acad. Sci., U.S.A.,93: 388-391, 1996; Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758,1997). Due to the very real possibility of inducing an anaphylacticreaction in patients with native allergen, most clinical therapists arequite hesitant to use high doses therapeutically and are thereforecompromising the likelihood of successful therapy.

[0180] In recent reports, recombinant technology has been used to reducethe allergenic potential of a major allergen without modifying the Tcell epitopes, and allowing higher doses of protein to be used intherapy (Tokai, T., et al., Nat. Biotechnol., 15(8): 754-758, 1997). Inaddition, a lack of understanding about the appropriate route ofadministration, the uncertainty of mechanisms responsible for inductionof allergy and the uncertainty of mechanisms by which immunotherapysuppresses or blocks the T cell-IgE-eosinophil/mast cell cycle havecontributed to the large number of equivocal studies and clinicaltrials. Recent studies in animal models dealing with mechanisms, routesof administration, adjuvants and vaccine formulations have increased thelikelihood that immunotherapy for allergies, including food allergies,will become a reproducibly successful clinical treatment when theappropriate therapeutic reagents are available (Sampson, H. A. andBurks, A. W., Annu. Rev. Nutr., 16: 161-177, 1996; Kaminogawa, S.,Biosci. Biotechnol. Biochem., 60(11): 1749-1756, 1996; Chapman, M. D.,et al., Allergy, 52: 374-379, 1997; Barbeau, W. E., Adv. Exp. Med.Biol., 415: 183-193, 1997; Cao, Y, et al., Immunology, 90(1): 46-51,1997; Garside, P. and Mowat, A. M., Crit. Rev. Immunol., 17(2): 119-137,1997; Rothe, M. J. and Grant-Kels, J. M., J. Am. Acad. Dermatol., 35(1):1-13, 1996; Strobel, S., Allergy, 50(20): 18-25, 1995; Kruisbeek, A. M.and Amsen, D., Curr. Opin. Immunol., 8(2): 233-244, 1996; Herz, U., etal., Adv. Exp. Med. Biol., 409: 25-32, 1996; Litwin, A., et al., J.Allergy Clin. Immunol., 100: 30-38, 1997; Vandewalker, M. L., Mo. Med.,94(7): 311, 1997; Marshall, G. D., Jr. and Davis, F., Nat. Biotechnol.,15(8): 718-719, 1997; Van Deusen, M. A., et al., Ann. Allergy AsthmaImmunol., 78: 573-580, 1997; Jacobsen, L., et al., Allergy, 52: 914-920,1997, Scheiner, O. and Kraft, D., Allergy 50(5): 384-391, 1995).

[0181] Relative to immunotherapy, the critical aspects of the modifiedpatatin genes described in this patent are that they can be used tosynthesize purified, deallergenized-protein which can be used forpatatin (potato) specific immunotherapy, with reduced potential foradverse and potentially fatal anaphylactic reactions in human orveterinary patients who have allergies to patatin or potatoes. Variousstrategies, including fixing or cross linking allergens, encapsulationof allergen for oral delivery, the use of small, T-cell epitope peptidesand most recently, the use of engineered recombinant proteins, ormodified gene vaccines are being tested in attempts to decrease thepotential for anaphylactic reactions while inducing tolerance (Cao, Y.,et al., Immunology, 90(1): 46-51, 1997; Chapman, M. D., et al., Allergy,52: 374-379, 1997; Chapman, M. D., et al., Int. Arch. Allergy Immunol.,113(1-3): 102-104, 1997; Collins, S. P., et al., Clin. Exp. Allergy,26(1): 36-42, 1996; Takai, T., et al., Mol. Immunol., 34(3): 255-261,1997; Takai, T., et al., Nat. Biotechnol., 15(8) 754-758, 1997;Jirapongsananruk, O. and Leung, D. Y. M., Ann. Allergy Asthma Immunol.,79: 5-20, 1997; Litwin, A., et al., J. Allergy Clin. Immunol., 100:30-38, 1997; Vandewalker, M. L., Mo. Med., 94(7): 311, 1997; Raz, E., etal., Proc. Natl. Acad. Sci., U.S.A., 93: 5141-5145, 1996; Hoyne, G. F.,et al., Clin. Immunol. Immunopathol., 80: S23-30, 1996; Hoyne, G. F., etal., Int. Immunol., 9(8): 1165-1173, 1997; Vrtala, S., et al., J. Clin.Invest., 99(7): 1673-1681, 1997; Sato, Y., et al., Science, 273:352-354, 1996; Lee, D. J., et al., Int. Arch. Allergy Immunol.,113(1-3): 227-230, 1997; Tsitoura, D. C., et al., J. Immunol., 157(5):2160-2165, 1996; Hsu, C. H., et al., Int. Immunol., 8(9):1405-1411,1996; Hsu, C. H., et al., Nat. Med., 2(5): 540-544, 1996).

[0182] The instant invention uses an engineered patatin protein, asexpressed in any living cell, with or without post-synthesismodifications, for immunotherapy by the routes of cutaneous orsubcutaneous exposure, injection, or by oral, gastro-intestinal,respiratory or nasal application, either with, or without the use ofspecific carriers, vehicles and adjuvants. The direct application ofnucleic acid encoding recombinant patatin as the in vivo (in thepatient) expression template (gene) as RNA-, DNA- or gene-vaccines isalso the intended use of the engineered genetic materials defined here,coding for patatin, but with modified IgE binding sites. It is also theintent of this patent to cover the use of these modified genes describedhere including insertion into various DNA vectors including adenovirus,retrovirus, pox virus and replicating or non-replicating eukaryoticexpression plasmids (Lee, D. J., et al., Int. Arch. Allergy Immunol.,113(1-3): 227-230, 1997) with various promoters and regulatorysequences, which can be inserted into the patient's somatic cells(dendritic cells, epithelial cells, muscle fiber-cells, fibroblasts,etc.) for the purpose of expressing the recombinant gene product toalter the patient's immune response to the patatin proteins (Lee D. J.,et al., Int. Arch. Allergy Immunol., 113(1-3): 227-230, 1997). Potentialroutes of administration foreseen in this application include previouslydescribed methods of encapsulation, emulsion, receptor or membranefusion mediated uptake and methods of direct permeabilization orinsertion of the DNA or corresponding RNA into the host cells.

[0183] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventors to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

EXAMPLES Example 1 Identification of Patatin as an Allergen

[0184] Since patatin is commonly obtained from an allergenic source(potato), it was hypothesized that patatins in fact encode an importantclass of offending potato allergens (patatin was reported as allergenicby Seppala, U. et al., J. Allergy Clin. Immunol. 103: 165-171, 1999).Assessment of potential allergens preferably include appropriate invitro testing for IgE binding, in this case with potato allergic sera(Fuchs, R. L. and Astwood, J. D., Food Technology, 50: 83-88, 1996;Astwood, J. D., et al., Monographs in allergy Vol. 32: Highlights infood allergy, pp. 105-120, 1996, Metcalfe, D. D., et al., CriticalReviews in Food Science and Nutrition, 36S: 165-186, 1996). It is therecommendation of a working group organized by the IFBC and the ILSIAllergy and Immunology Institute that proteins encoded by nucleic acidsequences from allergenic sources such as potato (a “less-commonly”allergenic source) should be examined for their ability to react withIgEs of potato-allergic patients using a minimum of five individualpatient sera (Metcalfe, D. D., et al., Critical Reviews in Food Scienceand Nutrition, 36S: 165-186, 1996). Patatin-17 protein was tested forIgE binding using standard in vitro testing with serum taken frompatients with bona fide well defined clinically displayed potato allergyas described below.

[0185] Clinical Characterization of Potato Allergic Subjects (SerumDonors)

[0186] Patients who suffer from potato allergy were identified at JohnsHopkins Clinic (Baltimore, Md.) and were evaluated for potato allergyusing clinical criteria outlined in Table 2.

[0187] Serum was obtained from patients with convincing clinical historyof potato allergy. The convincing history was defined as being one ormore of the following: a) positive potato allergic as evaluated bydouble-blind placebo-control food challenge b) anaphylaix and/orhospitalization due to the consumption of potatoes or c) dramatic skintest results. TABLE 2 Clinical patient data Flare/Wheal Patient ClinicalHistory (Skin prick test) DBPCFC (potato) HS01 Most recenthospitalization: Oct. 19, 1993  7/19, 4/14, 7/17 Not performed AD, A,AR, FH, MFS, IgE = 1397 KIAUa/L HS02 Most recent hospitalization: Jun.1994 20/26 Not performed AD, FH, Latex (+) RAST, MFS, IgE = 7544 K/LHS03 Most recent hospitalization: Jul. 27, 1995  5/13 Yes AD, A, FH,MFS, IgE = N/A HS05 Most recent hospitalization May 30, 1995  4/9 YesAD, A, FH, MFS, IgE = 12341 ng/ml HS06 Most recent hospitalization Jun.13, 1995  5/20, 4/13, 5/12 Yes AD, A FH, MFS IgE = N/A HS07 Not potatoallergic, allergic to egg, milk, High IgE control serum, not peanuts,seafood. AD, A, AR, FH, MFS allergic to potato. HS08 Non-atopic (normal)Low IgE control serum

Example 2 Western Blotting of Patatin Proteins

[0188] Western blotting experiments were performed using patatin proteinpurified to near homogeneity from corn plants genetically engineered toproduce patatin, patatin producing crude genetically engineered cornleaf extracts, crude potato tuber extracts, and non-transgenic corn leafsamples.

[0189] Protein samples were electrophoresed by SDS-PAGE (Laemmli, U. K.,Nature 227: 680-685, 1970) and were electroblotted onto nitrocellulose.Protein blots were processed by standard Western blotting(immunoblotting) techniques and were incubated in potato allergic serumdiluted 1:5 in PBS buffer for 1 hour. After washing the blots 3 timeswith PBS, the blots were incubated in biotinylated anti-IgE (JohnsHopkins Hospital, Baltimore, Md.) for 1 hour, followed by a 30 minuteincubation in HRP-linked avidin (Promega, New York, N.Y.). IgE-reactiveprotein bands were visualized by DAB staining (3,3 diaminobenzidine).The blots were dried and photographed. Individual blots are labeledaccording to patient serum used. As a control, one blot was incubated inanti-IgE only.

[0190] Patatins were shown to be an allergen of potato by examining thereactivity of purified patatin to sera obtained from patients allergicto potato. Sera from five potato allergic subjects were tested byWestern blotting techniques. All five sera reacted with purified patatinprotein.

[0191] Patatin isozymes (SEQ ID NOS:278-282, FIG. 1) were tested for IgEbinding by Western blotting. Isozymes of patatin were cloned into ayeast expression system and purified prior to analysis. The isozymeswere subjected to IgE western blotting as described above with theexception that all five patient sera were pooled. The resulting Westernblot of the yeast-expressed isozymes showed that all five isozymes boundIgE in a manner similar to patatin 17, and that all isozymes of patatintested are also allergens.

Example 3 Western Blotting of Patatin Proteins

[0192] Eighty-nine 10-mer peptides were synthesized using the GenosysSPOTs system, each consecutive 10-mer overlapping by 6 amino acids basedon the amino acid sequence of patatin 17 (SEQ ID NO:2). The peptideswere evaluated for IgE binding with five different potato allergicpatient sera using the same incubation procedures as described above.The results are summarized graphically in FIG. 2, showing major andminor allergenic epitopes. Interestingly, many of the immunogenicepitopes contain tyrosine. The peptide numbers, sequences, andimmunoreactivity is detailed in Table 3. TABLE 3 Peptide scan of patatin17 Peptide # (SEQ ID Cumulative NO) Peptide Sequence HS01 HS02 HS03 HS05HS06 Total  1 (16) QLGEMVTVLS 0.47 0.33 0.02 0.05 0.06 0.93  2 (17)MVTVLSIDGG 0.53 0.33 0.02 0.07 0.05 1  3 (18) LSIDGGGIRG 0.52 0.38 0.070.08 0.09 1.14  4 (19) GGGIRGIIPA 0.53 0.19 0.06 0.19 0.23 1.2  5 (20)RGIIPATILE 0.46 0.28 0.04 0.09 0.05 0.92  6 (21) PATILEFLEG 0.49 0.310.05 0.09 0.07 1.01  7 (22) LEFLEGQLQE 0.36 0.24 0.04 0.1 0.06 0.8  8(23) EGQLQEMDNN 0.29 0.19 0.02 0.09 0.05 0.64  9 (24) QEMDNNADAR 0.220.13 0.01 0.05 0.04 0.45 10 (25) NNADARLADY 0.21 0.17 0.03 0.05 0.070.53 11 (26) ARLADYFDVI 0.54 0.31 0.16 0.15 0.25 1.41 12 (27) DYFDVIGGTS0.61 0.34 0.46 0.06 0.15 1.62 13 (28) VIGGTSTGGL 0.63 0.72 0.05 0.150.09 1.64 14 (29) TSTGGLLTAM 0.3 0.17 0.03 0.06 0.09 0.65 15 (30)GLLTAMISTP 0.63 0.41 0.05 0.24 0.12 1.45 16 (31) AMISTPNENN 0.34 0.180.02 0.07 0.02 0.63 17 (32) TPNENNRPFA 0.46 0.22 0.03 0.19 0.07 0.97 18(33) NNRPFAAAKE 0.37 0.21 0.05 0.07 0.06 0.76 19 (34) FAAAKEIVPF 0.520.29 0.08 0.11 0.08 1.08 20 (35) KEIVPFYFEH 0.29 0.14 0.28 0.29 0.231.23 21 (36) PFYFEHGPQI 0.65 0.06 1.08 0.51 0.17 2.47 22 (37) EHGPQIFNPS0.34 0.15 0.03 0.05 0.06 0.63 23 (38) QIFNPSGQIL 0.33 0.29 0.02 0.070.07 0.78 24 (39) PSGQILGPKY 0 0 0.02 0 0.05 0.07 25 (40) ILGPKYDGKY 0 00.07 0 0.02 0.09 26 (41) KYDGKYLMQV 0.02 0 0.11 0.01 0.04 0.18 27 (42)KYLMQVLQEK 0.12 0.04 1.08 0.07 0.79 2.1 28 (43) QVLQEKLGET 0.46 0.160.01 0.07 0.02 0.72 29 (44) EKLGETRVHQ 0.5 0.12 0.01 0.07 0.04 0.74 30(45) ETRVHQALTE 0.42 0.16 0.03 0.05 0.03 0.69 31 (46) HQALTEVVIS 0.430.21 0.04 0.1 0.05 0.83 32 (47) TEVVISSFDI 0.44 0.25 0.05 0.08 0.04 0.8633 (48) ISSFDlKTNK 0.1 0.02 0.04 0.06 0.13 0.35 34 (49) DIKTNKPVIF 0.570.22 0.04 0.18 0.28 1.29 35 (50) NKPVIFTKSN 0 0.01 0.02 0.07 0.24 0.3436 (51) IFTKSNLANS 0 0 0.03 0.06 0.17 0.26 37 (52) SNLANSPELD 0.43 0.960.01 0.09 0.02 1.51 38 (53) NSPELDAKMY 0.18 0.12 0.01 0.05 0.05 0.41 39(54) LDAKMYDISY 0.54 0.26 0.19 0.15 0.23 1.37 40 (55) MYDISYSTAA 0.920.08 0.52 0.04 0.22 1.78 41 (56) SYSTAAAPTY 1.15 0.25 1.04 0.33 0.553.32 42 (57) AAAPTYFPPH 1.02 0.52 1.12 0.81 0.86 4.33 43 (58) TYFPPHYFVT0.02 0.01 0.54 0.03 0.24 0.84 44 (59) PHYFVTNTSN 0.03 0.01 1.17 0.130.44 1.78 45 (60) VTNTSNGDEY 0.23 0.15 0.04 0.03 0.03 0.48 46 (61)SNGDEYEFNL 0.33 0.25 0.08 0.1 0.11 0.87 47 (62) EYEFNLVDGA 0.34 0.250.07 0.1 0.2 0.96 48 (63) NLVDGAVATV 0.3 0.18 0.02 0.06 0.05 0.61 49(64) GAVATVADPA 0.45 0.54 0.01 0.07 0.02 1.09 50 (65) TVADPALLSI 0.480.29 0.01 0.07 0.03 0.88 51 (66) PALLSISVAT 0.65 0.33 0.02 0.1 0.01 1.1152 (67) SISVATRLAQ 0.61 0.23 0.14 0.53 0.53 2.04 53 (68) ATRLAQKDPA 0.870.34 0.05 0.29 0.22 1.77 54 (69) AQKDPAFASI 0.86 0.32 0.04 0.12 0.031.37 55 (70) PAFASIRSLN 0.81 0.15 0.05 0.51 0.59 2.11 56 (71) SIRSLNYKKM0.07 0.01 0.17 0.07 0.11 0.43 57 (72) LNYKKMLLLS 0.05 0.01 0.35 0.080.39 0.88 58 (73) KMLLLSLGTG 1.15 0.15 0.04 0.38 0.71 2.43 59 (74)LSLGTGTTSE 0.34 0.23 0.02 0.04 0.03 0.66 60 (75) TGTTSEFDKT 0.92 0.390.6 0.1 0.09 2.1 61 (76) SEFDKTYTAK 1.33 1.35 1.41 0.12 0.28 4.49 62(77) KTYTAKEAAT 1.36 0.94 1.11 0.76 0.4 4.57 63 (78) AKEAATWTAV 0.450.15 0.01 0.2 0.04 0.85 64 (79) ATWTAVHWML 0.1 0.02 0.01 0.08 0.06 0.2765 (80) AVHWMLVIQK 0.69 0.05 0.03 0.43 0.62 1.82 66 (81) MLVIQKMTDA 0.320.15 0.02 0.15 0.03 0.67 67 (82) QKMTDYYLST 0.26 0.125 0.03 0.21 0.050.675 68 (83) DAASSYMTDY 0.2 0.14 0.08 0.08 0.1 0.6 69 (84) SYMTDYYLST0.5 0.03 0.32 0.06 0.11 1.02 70 (85) DYYLSTAFQA 0.14 0 0.22 0.03 0.130.52 71 (86) STAFQALDSK 0.4 0.3 0.04 0.06 0.08 0.88 72 (87) QALDSKNNYL0.44 0.46 0.28 0.26 0.43 1.87 73 (88) SKNNYLRVQE 0.44 0.05 1.31 0.070.21 2.08 74 (89) YLRVQENALT 1.38 0.03 1.31 0.11 0.2 3.03 75 (90)QENALTGTTT 0.47 0.25 0 0.06 0 0.78 76 (91) LTGTTTEMDD 0.41 0.24 0 0.06 00.71 77 (92) TTEMDDASEA 0.38 0.3 0 0.05 0 0.73 78 (93) DDASEANMEL 0.440.24 0 0.06 0 0.74 79 (94) EANMELLVQV 0.42 0.27 0 0.04 0 0.73 80 (95)ELLVQVGENL 0.4 0.25 0 0.05 0 0.7 81 (96) QVGENLLKKP 0.44 0.14 0 0.07 00.65 82 (97) NLLKKPVSED 0.47 0.2 0 0.03 0 0.7 83 (98) KPVSEDNPET 0.270.21 0 0.03 0 0.51 84 (99) EDNPETYEEA 0.13 0.11 0 0.01 0 0.25 85 (100)ETYEEALKRF 1.26 1.2 1.36 0.53 0.71 5.06 86 (101) EALKRFAKLL 1.38 0.04 01.06 1.12 3.6 87 (102) RFAKLLSDRK 0.98 0.05 0 0.84 0.94 2.81 88 (103)LLSDRKKLRA 0.2 0.01 0 0.37 0.51 1.09 89 (104) RKKLRANKAS 0.28 0 0 0.310.64 1.23 Patient 41.84 20.565 18.1 14.17 16.55 Cumulative Totals

Example 4 Identification of Result Effective Substitutions

[0193] For each major and several minor allergenic epitopes of patatin,result effective substitutions were identified by synthesizing peptidesthat were altered by individually substituting an alanine residue ateach non-alanine position in the epitope. Similarly, the reportednucleic acid sequence encoding corn patatin (U.S. Pat. No. 5,882,668;clone 5c9) was evaluated for IgE binding by producing peptides atcorresponding positions to the potato patatin protein.

[0194] For example, Epitope 41 was analyzed by alanine scanning andrational substitution as follows. Epitope 41 SEFDKTYTAK (SEQ ID NO:76)Alanine scan AEFDKTYTAK (SEQ ID NO:165) SAFDKTYTAK (SEQ ID NO:166)SEADKTYTAK (SEQ ID NO:167) SEFAKTYTAK (SEQ ID NO:168) SEFDATYTAK (SEQ IDNO:169) SEFDKAYTAK (SEQ ID NO:170) SEFDKTATAK (SEQ ID NO:171) SEFDKTYAAK(SEQ ID NO:172) SEFDKTYTAA (SEQ ID NO:173) Rational substitutionAFFDKTYTAK (SEQ ID NO :283) SEFDKTFTAK (SEQ ID NO:176) Corn homologCIFDSTYTAK (SEQ ID NO:284)

[0195] Selected epitopes were analyzed by alanine scanning and rationalsubstitution. Immunoassay with potato-allergic serum was used asdescribed above. Table 4 summarizes the results of these experiments toidentify result effective substitutions for patatin. Blank spaces in thetable indicate that binding of the peptide to patient IgE was notdetectable. TABLE 4 Scanning of patatin for result effectivesubstitutions Binding of modified peptides by patient IgE as measured byOD Sequence SEQ ID NO HS03 HS06 HS01 HS02 DYFDVIGGTS 105 0.12 0.16 0.36DYFDVIAGTS 106 0.14 0.17 0.4 VIGGTSTGGL 107 0.04 VIAGTSTGAL 108AFYFEHGPQI 109 0.96 0.5 0.78 PAYFEHGPQI 110 0.75 0.41 0.69 PFAFEHGPQI111 PFYAEHGPQI 112 0.7 0.43 0.79 PFYFAHGPQI 113 0.93 1.07 0.59 1.44PFYFEAGPQI 114 0.08 0.93 0.65 1.34 PFYFEHAPQI 115 0.75 0.54 1.11PFYFEHGAQI 116 0.63 0.29 0.6 PFYFEHGPAI 117 0.63 0.25 0.56 PFYFEHGPQA118 0.27 0.16 0.33 TFYLENGPKI 119 0.05 0.48 0.68 1.07 PFFFEHGPQI 120AYLMQVLQEK 121 0.26 0.11 0.53 KALMQVLQEK 122 KYAMQVLQEK 123 0.11 0.430.1 1.25 KYLAQVLQEK 124 0.22 0.48 0.11 1.34 KYLMAVLQEK 125 0.22 0.830.16 1.33 KYLMQALQEK 126 0.11 0.6 0.15 0.95 KYLMQVAQEK 127 0.53 0.150.81 KYLMQVLAEK 128 0.06 0.69 0.11 1.34 KYLMQVLQAK 129 0.74 0.79 0.050.58 KYLMQVLQEA 130 0.28 0.27 0.37 VFLHDKIKSL 131 0.06 0.26 0.41AYSTAAAPTY 132 0.1 0.12 0.12 SASTAAAPTY 133 SYATAAAPTY 134 0.16 0.130.37 SYSAAAAPTY 135 0.13 0.12 0.32 SYSTAAAATY 136 0.15 0.13 0.34SYSTAAAPAY 137 0.15 0.14 0.29 SYSTAAAPTA 138 0.55 0.54 1.13 CISTSAAPTY139 0.4 SYSTAAAPAF 140 0.39 1.02 0.65 1.42 AFAAAAAPTY 141 0.07SYSTAAAPTF 142 0.15 0.97 0.48 1.09 STSAAPTYFP 143 0.21 0.23 0.39STSAAPTFFP 144 0.23 STSAAPTAFP 145 0.08 STAAAPTFFP 146 0.12 0.28AAAATYFPPH 147 0.13 0.1 0.05 AAAPAYFPPH 148 0.07 0.04 AAAPTAFPPH 149AAAPTYAPPH 150 0.23 0.14 0.21 AAAPTYFAPH 151 0.45 0.18 0.44 AAAPTYFPAH152 0.15 0.07 0.18 AAAPTYFPPA 153 0.1 0.06 0.31 SAAPTYFPAH 154 0.77 0.730.96 AAAPAFFPPH 155 AAAPPFFPPH 156 AAAPTFFPPH 157 SISVATRLAQ 158 0.260.26 AMSMLTKEVH 159 PAFASIRSLN 160 PNFNAGSPTE 161 KMLLLSLGTG 162NYLIISVGTG 163 0.49 1.08 0.64 1.48 KMLLLSLGAG 164 0.13 AEFDKTYTAK 1650.09 0.22 1.34 SAFDKTYTAK 166 0.66 0.71 0.06 1.42 SEADKTYTAK 167 0.99SEFAKTYTAK 168 0.5 0.57 0.91 SEFDATYTAK 169 0.17 SEFDKAYTAK 170 0.1 0.241.38 SEFDKTATAK 171 0.81 SEFDKTYAAK 172 0.2 0.35 1.39 SEFDKTYTAA 173 0.11.18 KQAEKYTAEQ 174 0.08 0.24 SEFDAAFAAA 175 SEFDKTFTAK 176 0.09 0.160.07 1.45 AEKYTAEQCA 177 ATYTAKEAAT 178 0.24 0.18 KAYTAKEAAT 179 0.280.33 KTATAKEAAT 180 KTYAAKEAAT 181 0.1 0.32 0.73 KTYTAAEAAT 182 0.35KTYTAKAAAT 183 0.4 0.59 0.82 KTYTAKEAAA 184 0.36 EKYTAEQCAK 185AAFAAAEAAT 186 KTFTAKEAAT 187 QALHCEKKYL 188 QALDSKAAYL 189 QALDSKNNFL190 QALHCENNFL 191 CEKKYLRIQD 192 1.01 0.16 SKNNFLRVQE 193 SENNYLRVQE194 0.31 0.96 0.42 1 ALRVQENALT 195 YARVQENALT 196 1.06 1.02 0.05 0.54YLAVQENALT 197 0.37 1.04 0.11 1.06 YLRAQENALT 198 1.1 1 0.06 1.26YLRVAENALT 199 1.03 0.92 0.08 1.26 YLRVQANALT 200 1.05 0.92 0.06 1.24YLRVQEAALT 201 0.93 0.92 0.07 1.11 YLRVQENAAT 202 0.94 0.93 0.04 1.24YLRVQENALA 203 1.05 0.96 0.43 1.16 YLRIQDDTLT 204 1.07 0.85 0.39 1.12YLTVAAAALT 205 1.05 0.86 0.28 1.33 FLRVQENALT 206 NNYLRVQENA 207 0.230.88 0.5 1.17 KKYLRIQDDT 208 0.26 0.09 0.37 NNFLRVQENA 209 NAYLRVQENA210 0.17 1.02 0.53 1.06 ATYEEAKLRF 211 0.26 1.03 0.65 EAYEEALKRF 2120.06 0.43 0.33 ETAEEALKRF 213 1.04 ETYAEALKRF 214 0.62 1.02 1.15ETYEAALKRF 215 1.06 0.38 0.89 ETYEEAAKRF 216 0.08 0.1 0.9 ETYEEALARF 2170.11 ETYEEALKAF 218 0.1 ETYEEALKRA 219 0.1 GTNAQSLADF 220 ETYEAALAAF 2210.07 0.78 0.33 0.77 ETFEEALKRF 222 YEEALKTFAK 223 1.08 0.85 0.14 1.46FEEALKRFAK 224 0.46 0.72 0.67 AALKRFAKLL 225 0.15 0.17 EAAKRFAKLL 2260.08 0.33 0.05 EALARFAKLL 227 0.09 EALKAFAKLL 228 EALKRAAKLL 229 0.080.07 EALKRFAALL 230 EALKRFAKAL 231 0.06 0.09 0.1 EALKRFAKLA 232 0.06 0.1QSLADFAKQL 233 AALAAFAKLL 234 LADFAKQLSD 235 DFAKQLSDER 236 0.17AFAALLSDRK 237

[0196] Result effective substitutions were identified by a reduction inIgE binding ability with respect to the non-substituted peptidesequence. Table 5 shows the identified result effective substitutions.Blank spaces in the table indicate that binding of the peptide topatient IgE was not detectable. Many substitutions of alanine orphenylalanine for the original tyrosine resulted in reduced oreliminated antibody binding. TABLE 5 Result effective substitutions ofpatatin Location (SEQ ID NO) Peptide (SEQ ID NO) HS03 HS06 HS01 HS02Minor PFYFEHGPQI  (36) 1.08 0.17 0.65 0.06 Epitope ::A::::::: (111) 21::F::::::: (r) (120) :::::::::A (118) 0.27 0.16 0.33 Minor KYLMQVLQEK (42) 1.08 0.79 0.12 0.04 Epitope :A:::::::: (122) 27 :::::::::A (130)0.28 0.27 0.37 VFLHDKIKSL (c) (131) 0.06 0.26 0.41 Major SYSTAAAPTY (56) 1.04 0.55 1.15 0.25 Epitope A::::::::: (132) 0.1 0.12 0.12 41:A:::::::: (133) AFAA:::::: (r) (141) 0.007 CI::S::::: (c) (139) 0.04Overlap STAAAPTYFP (238) Epitope ::S::::A:: (r) (145) 0.08 41/42 MajorAAAPTYFPPH  (57) 1.12 0.86 1.02 0.52 Epitope ::::A::::: (148) 0.07 0.0442 :::::A:::: (149) (57) ::::AF:::: (r) (155) ::::PF:::: (r) (156):::::F:::: (r) (157) Major SEFDKTYTAK  (76) 0.12 0.28 1.33 1.35 Epitope::::A::::: (169) 0.17 61 KQAE:YTAEQ (c) (174) 0.08 0.24 ::::AAFA:A (r)(175) Major KTYTAKEAAT  (77) 1.11 0.04 1.36 0.94 Epitope A:::::::::(178) 0.24 0.18 62 ::A::::::: (180) :::::A:::: (182) 0.35 AAFA:A:::: (r)(186) ::F::::::: (r) (187) EK:::EQC:K (c) (185) Minor QALDSKNNYL  (87)0.28 0.43 0.44 0.46 Epitope :::HCEKK:: (c) (188) 72 ::::::AA:: (r) (189)::::::::F: (r) (190) :::::E::F: (r) (240) Minor SKNNYLRVQE  (88) 1.310.21 0.44 0.05 epitope ::F::::::: (r) (193) 73 Minor YLRVQENALT  (87)1.31 0.2 1.38 0.03 epitope A::::::::: (195) 74 F::::::::: (r) (206)Overlap NNYLRVQENA (207) 0.23 0.88 0.5 1.17 epitope ::F::::::: (r) (209)73/74 Major ETYEEALKRF (100) 1.36 0.71 1.26 1.2 epitope :::::::A:: (217)0.11 85 ::::::::A: (218) 0.1 :::::::::A (219) 0.1 ::F::::::: (r) (222)G:NAQS:AD: (c) (220) Major EALKRFAKLL (101) 0 1.12 1.38 0.04 Epitope:::A:::::: (227) 0.09 86 ::::A::::: (228) :::::A:::: (229) 0.08 0.07:::::::A:: (230) ::::::::A: (231) 0.06 0.09 :::::::::A (232) 0.06SD:AD:::Q: (c) (241) A::AA::::: (r) (234) Epitope LKRFAKLLSD (239)overlap (NO 86/87 BINDING) Major RFAKLLSDRK (102) 0 0.94 0.98 0.05Epitope D:::Q:::ER (c) (236) 0.17 87 A::A:::::: (r) (237)

Example 5 Site Directed Mutagenesis

[0197] To introduce site specific mutations, the cloned DNA sequence ofpatatin (SEQ ID NO:1 encoding patatin protein SEQ ID NO:2; pMON 26820)was subjected to PCR with primers SEQ ID NO:3 and SEQ ID NO:4 toincorporate part of the α-factor signal sequence (Pichia expressionmanual, Invitrogen, Carlsbad, Calif.), and EcoRI and XhoI restrictionsites to facilitate cloning into the Pichia pastoris yeast secretionvector pPIC9 (GenBank accession number Z46233; Invitrogen, Carlsbad,Calif.). Typical PCR conditions are 25 cycles 94° C. denaturation for 1minute, 45° C. annealing for one minute and 72° C. extension for 2minutes; plus one cycle 72° C. extension for 10 minutes. A 50 μLreaction contains 30 pmol of each primer and 1 μg of template DNA; and1×PCR buffer with MgCl2, 200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μMdCTP, 2.5 units of Pwo DNA polymerase. PCR reactions are performed inRoboCycler Gradient 96 Temperature Cycler (Stratagene, La Jolla,Calif.).

[0198] The amplified fragment SEQ ID NO:5 was digested with restrictionenzymes XhoI and EcoRI and cloned into the pBluescript vector(Stratagene, La Jolla, Calif.), digested with the same two restrictionenzymes. The resulting plasmid (pMON 26869) was used foroligonucleotide-directed mutagenesis using the Bio-Rad mutagenesis kitbased on the method of Kunkel (Proc. Natl. Acad. Sci. U.S.A., 82:477-492, 1985). Briefly, single-stranded pMON26869 was used as templatefor mutagenesis and was prepared by superinfection of plasmid containingcells with M13K07 (Gorman, et al., DNA Prot. Eng. Techniques, 2: 3-10,1990). The mutagenic oligonucleotides are SEQ ID NOS:8-15 (reversecomplement). DNA purified from transformed DH5αa E. coli colonies wasused for sequence determination. Sequencing was performed using the ABIPRISM sequencing kit (Perkin Elmer Biosystems, Foster City, Calif.). Theresulting plasmid containing the mutation in the patatin gene wasdigested with restriction enzymes XhoI and EcoRI.

[0199] The patatin nucleic acid fragment was then ligated into the pPIC9vector (Invitrogen, Carlsbad, Calif.), digested with the same tworestriction enzymes to afford plasmid pMON37401. Pichia pastoris KM71cells were electroporated with pMON37401 containing the appropriatemutation. The resulting transformed cells were used to produce proteinin Pichia pastoris using the procedure supplied by the manufacturer(Invitrogen, Carlsbad, Calif.). The encoded protein contains an alphafactor signal cleavage site. Plasmid pMON37401 encodes SEQ ID NO:6 whichis cleaved to afford SEQ ID NO:7, having four amino acids added at theN-terminus of amino acids 24-386 of SEQ ID NO:2. Position four of SEQ IDNO:7 therefore corresponds to position 23 of SEQ ID NO:2.

[0200] The concentration of patatin in the culture was determined usinga patatin ELISA assay and the enzyme activity was measured using themethod of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). Thevariants containing multiple mutations were further purified using MonoQ and hydrophobic interaction chromatography (HIC). Each culture waspurified by first sizing on Amicon YM10 membranes (Millipore, Bedford,Mass.) to a >10 kDa fraction, followed by chromatography on the Mono QHR 10/10 column (Pharmacia, Piscataway, N.J.). For chromatography on theMono Q column, the samples were loaded on the column in 25 mM Tris pH7.5 and eluted with a gradient of 1.0 M KCl in 25 mM Tris pH 7.5.Fractions containing patatin protein were determined using SDS-PAGE. Forchromatography on the HIC column, the appropriate fractions were pooledand dialyzed into 1 M ammonium sulfate in 25 mM Tris pH 7.5. Thedialyzed sample was then loaded on 16/10 phenyl Sepharose column(Pharmacia, Piscataway, N.J.) and eluted with a gradient of 25 mM TrispH7.5.

[0201] The protein concentration was determined using the Bradfordmethod, using BSA as a standard. SDS-PAGE analysis showed that theseproteins were essentially pure. The esterase activity of the newlyformed variants are shown in Table 6. The activity was determined usingp-nitrophenyl caprate substrate as described by Hofgen and Willmitzer(Plant Science, 66: 221-230, 1990). TABLE 6 Esterase activity of patatinmutants Activity Variant (mOD · min⁻¹μg⁻¹) Wild type 93.2 Y106F 51.1Y129F 74.7 Y185F 85.6 Y193F 82.2 Y185F/Y193F 99.4 Y270F 163.4 Y316F94.88 Y362F 130.7 Y106F/Y129F/Y185F/Y193F/Y270F/Y316F/Y362F 57.1Y185F/Y193F/Y270F/Y316F/Y362F 161.5

[0202] Patatin proteins having a phenylalanine substitution at each ofthe amino acid positions 106, 129, 185, 193, 270, 316 and 362 (numberscorrespond to positions in SEQ ID NO:2) of expressed SEQ ID NO:7 exhibitfull enzyme activity. Proteins having multiple substitutions alsodisplayed full enzyme activity.

[0203] In addition to nucleotide sequences encoding conservative aminoacid changes within the fundamental polypeptide sequence, biologicallyfunctional equivalent nucleotide sequences include nucleotide sequencescontaining other base substitutions, additions, or deletions. Theseinclude nucleic acids containing the same inherent genetic informationas that contained in the cDNA which encode peptides, polypeptides, orproteins conferring pathogen resistance the same as or similar to thatof pathogen upon host cells and plants. Such nucleotide sequences can bereferred to as “genetically equivalent modified forms” of the cDNA, andcan be identified by the methods described herein.

[0204] Mutations made in the cDNA, plasmid DNA, genomic DNA, syntheticDNA, or other nucleic acid encoding the deallergenized gene preferablypreserve the reading frame of the coding sequence. Furthermore, thesemutations preferably do not create complementary regions that couldhybridize to produce secondary mRNA structures, such as loops orhairpins, that would adversely affect mRNA translation.

[0205] Although mutation sites can be predetermined, it is not necessarythat the nature of the mutations per se be predetermined. For example,in order to select for optimum characteristics of mutants at a givensite, random mutagenesis can be conducted at the target codon.

[0206] Alternatively, mutations can be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native cDNAsequence. Following ligation, the resulting reconstructed nucleotidesequence encodes a derivative form having the desired amino acidinsertion, substitution, or deletion.

Example 6 Construction of Permutein Sequences

[0207] Nucleic acid sequences encoding permutein proteins havingrearranged N-terminus/C-terminus protein sequences can be made byfollowing the general method described by Mullins et al. (J. Am. Chem.Soc. 116: 5529-5533, 1994). The steps are shown in FIG. 3. The Figureand the following Examples involve the design and use of a linker regionseparating the original C-terminus and N-terminus, but the use of alinker is not a critical or required element of permutein design.

[0208] Two sets of oligonucleotide primers are used in the constructionof a nucleic acid sequence encoding a permutein protein. In the firststep, oligonucleotide primers “new N-termini” and “linker start” areused in a PCR reaction to create amplified nucleic acid molecule “newN-termini fragment” that contains the nucleic acid sequence encoding thenew N-terminal portion of the permutein protein, followed by thepolypeptide linker that connects the C-terminal and N-terminal ends ofthe original protein. In the second step, oligonucleotide primers “newC-termini” and “linker end” are used in a PCR reaction to createamplified nucleic acid molecule “new C-termini fragment” that containsthe nucleic acid sequence encoding the same linker as used above,followed by the new C-termini portion of the permutein protein. The “newN-termini” and “new C-termini” oligonucleotide primers are designed toinclude appropriate restriction enzyme recognition sites which assist inthe cloning of the nucleic acid sequence encoding the permutein proteininto plasmids.

[0209] Any suitable PCR conditions and polymerase can be used. It isdesirable to use a thermostable DNA polymerase with high fidelity toreduce or eliminate the introduction of sequence errors. Typical PCRconditions are 25 cycles 94° C. denaturation for 1 minute, 45° C.annealing for one minute and 72° C. extension for 2 minutes; plus onecycle 72° C. extension for 10 minutes. A 50 μL reaction contains 30 pmolof each primer and 1 μg of template DNA; and 1×PCR buffer with MgCl₂,200 μM dGTP, 200 μM dATP, 200 μM dTTP, 200 μM dCTP, 2.5 units of Pwo DNApolymerase. PCR reactions are performed in RoboCycler Gradient 96Temperature Cycler (Stratagene, La Jolla, Calif.).

[0210] The amplified “new N-termini fragment” and “new C-terminifragment” are annealed to form a template in a third PCR reaction toamplify the full-length nucleic acid sequence encoding the permuteinprotein. The DNA fragments “new N-termini fragment” and “new C-terminifragment” are resolved on a 1% TAE gel, stained with ethidium bromide,and isolated using the QIAquick Gel Extraction Kit (Qiagen, Valencia,Calif.). These fragments are combined in equimolar quantities witholigonucleotide primers “new N-termini” and “new C-termini” in the thirdPCR reaction. The conditions for the PCR are the same as usedpreviously. PCR reaction products can be purified using the QIAquick PCRpurification kit (Qiagen, Valencia, Calif.).

[0211] Alternatively, a linker sequence can be designed containing arestriction site, allowing direct ligation of the two amplified PCRproducts.

Example 7 Construction of Plasmid pMON 37402

[0212] The patatin protein contains a trypsin protease sensitive site atthe arginine amino acid at position 246, as determined byelectrophoresis of a trypsin digest reaction. In order to determine ifthe exposed protease site is an antigenic epitope, a permutein wasconstructed using positions 246-247 as a breakpoint.

[0213] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37402 was created using the method illustrated in FIG. 3and described in Example 6. Nucleic acid molecule “new N-terminifragment” was created and amplified from the sequence encoding patatinin plasmid pMON26820 using oligonucleotide primers 27 (SEQ ID NO:242)and 48 (SEQ ID NO:243). Nucleic acid molecule “new C-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 36 (SEQID NO:245). The full-length nucleic acid molecule encoding the permuteinprotein was created and amplified from annealed fragments “new N-terminifragment” and “new C-termini fragment” using oligonucleotide primers 27(SEQ ID NO:242) and 36 (SEQ ID NO:245).

[0214] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0215] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON 37402 (containing SEQ IDNO:246, encoding protein sequence SEQ ID NO:247).

Example 8 Construction of Plasmid pMON 37405

[0216] Amino acids 201-202, near tyrosine 193, were chosen as abreakpoint for the construction of a permutein protein.

[0217] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37405 was created using the method illustrated in FIG. 3and described in Example 6. Nucleic acid molecule “New N-terminifragment” was created and amplified from the sequence encoding patatinin plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243)and 58 (SEQ ID NO:249). Nucleic acid molecule “New C-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 59 (SEQID NO:249). The full-length nucleic acid molecule encoding the permuteinprotein was created and amplified from annealed fragments “New N-terminifragment” and “New C-termini fragment” using oligonucleotide primers 58(SEQ ID NO:248) and 59 (SEQ ID NO:249).

[0218] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0219] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON 37405 (containing SEQ IDNO:250, encoding protein sequence SEQ ID NO:251).

Example 9 Construction of Plasmid pMON 37406

[0220] Amino acids 183-184, adjacent to tyrosine 185, were chosen as abreakpoint for the construction of a permutein protein.

[0221] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37406 was created using the method illustrated in FIG. 3and described in Example 6. Nucleic acid molecule “New N-terminifragment” was created and amplified from the sequence encoding patatinin plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243)and 60 (SEQ ID NO:252). Nucleic acid molecule “New C-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 61 (SEQID NO:253). The full-length nucleic acid molecule encoding the permuteinprotein was created and amplified from annealed fragments “New N-terminifragment” and “New C-termini fragment” using oligonucleotide primers 60(SEQ ID NO:252) and 61 (SEQ ID NO:253).

[0222] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0223] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON37406 (containing SEQ IDNO:254, encoding protein sequence SEQ ID NO:255).

Example 10 Construction of Plasmid pMON 37407

[0224] Amino acids 268-269, adjacent to tyrosine 270, were chosen as abreakpoint for the construction of a permutein protein.

[0225] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37407 was created using the method illustrated in FIG. 3and described in Example 6. Nucleic acid molecule “New N-terminifragment” was created and amplified from the sequence encoding patatinin plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243)and 62 (SEQ ID NO:256). Nucleic acid molecule “New C-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 63 (SEQID NO:257). The full-length nucleic acid molecule encoding the permuteinprotein was created and amplified from annealed fragments “New N-terminifragment” and “New C-termini fragment” using oligonucleotide primers 62(SEQ ID NO:256) and 63 (SEQ ID NO:257).

[0226] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0227] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON37407 (containing SEQ IDNO:258, encoding protein sequence SEQ ID NO:259).

Example 11 Construction of Plasmid pMON 37408

[0228] Amino acids 321-322, near tyrosine 216, were chosen as abreakpoint for the construction of a permutein protein.

[0229] The nucleic acid sequence encoding the permutein protein inplasmid pMON 37408 was created using the method illustrated in FIG. 3and described in Example 6. Nucleic acid molecule “New N-terminifragment” was created and amplified from the sequence encoding patatinin plasmid pMON26820 using oligonucleotide primers 48 (SEQ ID NO:243)and 64 (SEQ ID NO:260). Nucleic acid molecule “New C-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON26820 using oligonucleotide primers 47 (SEQ ID NO:244) and 65 (SEQID NO:261). The full-length nucleic acid molecule encoding the permuteinprotein was created and amplified from annealed fragments “New N-terminifragment” and “New C-termini fragment” using oligonucleotide primers 64(SEQ ID NO:260) and 65 (SEQ ID NO:261).

[0230] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases XhoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). Plasmid pMON26869 (derivative of pPIC9, Invitrogen, Carlsbad, Calif.) was digestedwith restriction endonucleases XhoI and EcoRI, and gel purified,resulting in an approximately 2900 base pair vector fragment. Thepurified restriction fragments were combined and ligated using T4 DNAligase.

[0231] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON37408 (containing SEQ IDNO:262, encoding protein sequence SEQ ID NO:263).

Example 12 Production of Permutein Proteins in Pichia pastoris

[0232] Plasmids pMON37402, pMON37405, pMON37406, pMON37407, andpMON37408 were individually used to electroporate KM71 cells from Pichiapastoris according to the procedure supplied by the manufacturer(Invitrogen, Carlsbad, Calif.). The resulting transformed cells wereused to produce protein in Pichia pastoris following the proceduresupplied by the manufacturer (Invitrogen, Carlsbad, Calif.).

[0233] The concentration of patatin in the culture was determined usinga patatin ELISA assay and the enzyme activity was measured using themethod of Hofgen and Willmitzer (Plant Science, 66: 221-230, 1990). Thevariants containing multiple mutations were further purified using MonoQ and hydrophobic interaction chromatography (HIC). Each culture waspurified by first sizing on YM10 membranes (Amicon, MA) to a [>10 kDa]fraction, followed by chromatography on the Mono Q HR 10/10 column(Pharmacia, NJ). For chromatography on the Mono Q column, the sampleswere loaded on the column in 25 mM Tris pH 7.5 and eluted with agradient of 1.0 M KCl in 25 mM Tris pH 7.5. Fractions containing patatinprotein were determined using SDS-PAGE. For chromatography on the HICcolumn, the appropriate fractions were pooled and dialyzed into 1 Mammonium sulfate in 25 mM Tris pH 7.5. The dialyzed sample was thenloaded on 16/10 phenyl Sepharose column (Pharmacia, NJ) and eluted witha gradient of 25 mM Tris pH7.5.

[0234] The protein concentration was determined using the Bradfordmethod, using BSA as a standard. SDS-PAGE analysis showed that theseproteins were essentially pure. The esterase activity of the variantsare shown in Table 7. TABLE 7 Activity of permuteins pMON BreakpointActivity (ΔOD min⁻¹μg⁻¹) Native enzyme  83.21 pMON37402 246/247 66.7pMON37405 201/202 No expression pMON37406 183/184 No expressionpMON37407 268/269 12.1 pMON37408 321/322 No expression

[0235] The activity was determined using p-nitrophenyl caprate substrateas described by Hofgen and Willmitzer (Plant Science, 66: 221-230,1990).

Example 13 Insect Bioefficacy Assays

[0236] Assays for activity against larvae of SCRW are carried out byoverlaying the test sample on an agar diet similar to that described byMarrone (J. Econ. Entom. 78: 290-293, 1985). Test samples were preparedin 25 mM Tris, pH 7.5 buffer. Neonate larvae are allowed to feed on thetreated diet at 26° C., and mortality and growth stunting were evaluatedafter 5 or 6 days. The results of this assay are shown in Table 8. TABLE8 Insect bioefficacy assay Protein (200 ppm) Mean Survival Weight %Weight Reduction Tris buffer (control) 1.26 ± 0.3  — Wild Type 0.21 ±0.02 83 pMON37402 0.21 ± 0.03 83 pMON37407 0.32 ± 0.04 75

[0237] These data demonstrate that the growth of the SCRW larvae issimilarly reduced upon ingestion of the proteins encoded by pMON37402and pMON37407 as compared to the wild type patatin protein.

Example 14 Permutein Sequences Improved for Monocot Expression

[0238] Modification of coding sequences has been demonstrated above toimprove expression of insecticidal proteins. A modified coding sequencewas thus designed to improve expression in plants, especially corn (SEQID NO:264).

Example 15 Construction of pMON40701 for Monocot Expression

[0239] Plasmid pMON19767 was digested with restriction endonucleasesNcoI and EcoRI and the 1100 bp gene fragment was purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The two purified restriction fragments were combined andligated using T4 DNA ligase.

[0240] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON40700. Plasmid pMON40700 wasdigested with restriction endonuclease NotI and the resulting 2200 bpDNA fragment was purified using the QIAquick PCR purification kit(Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested withrestriction endonuclease NotI, and gel purified, resulting in anapproximately 4200 base pair vector fragment. The two purifiedrestriction fragments were combined and ligated using T4 DNA ligase.

[0241] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on kanamycin-containing plates. The resultingplasmid was designated pMON40701 (containing SEQ ID NO:264, encodingprotein sequence SEQ ID NO:265).

Example 16 Construction of pMON40703 for Monocot Expression

[0242] The nucleic acid sequence encoding the permutein protein inplasmid pMON40703 was created using the method illustrated in FIG. 3 anddescribed in Example 6. Nucleic acid molecule “New N-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn1 (SEQ ID NO:266) and Syn2(SEQ ID NO:267). Nucleic acid molecule “New C-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn4(SEQ ID NO:269). The full-length nucleic acid molecule encoding thepermutein protein was created and amplified from annealed fragments “NewN-termini fragment” and “New C-termini fragment” using oligonucleotideprimers Syn1 (SEQ ID NO:266) and Syn4 (SEQ ID NO:269).

[0243] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases NcoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

[0244] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON40702. Plasmid pMON40702 wasdigested with NotI, and the resulting 2200 bp DNA fragment was purifiedusing the QIAquick PCR purification kit (Qiagen, Valencia, Calif.).Plasmid pMON30460 was digested with restriction endonuclease NotI, andgel purified, resulting in an approximately 4200 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

[0245] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on kanamycin-containing plates. The resultingplasmid was designated pMON40703 (containing SEQ ID NO:270, encodingprotein sequence SEQ ID NO:271). Plasmid pMON40703 encodes a permuteinprotein with a “breakpoint” at positions 246/247 of the wild typepatatin protein sequence (SEQ ID NO:2). The first 23 amino acids of SEQID NO:2 are a signal peptide sequence which is cleaved in the matureprotein.

Example 17 Construction of pMON40705 for Monocot Expression

[0246] The nucleic acid sequence encoding the permutein protein inplasmid pMON40705 was created using the method illustrated in FIG. 3 anddescribed in Example 6. Nucleic acid molecule “New N-termini fragment”was created and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn10 (SEQ ID NO:272) and Syn2(SEQ ID NO:267). Nucleic acid molecule “New C-termini fragment” wascreated and amplified from the sequence encoding patatin in plasmidpMON19767 using oligonucleotide primers Syn3 (SEQ ID NO:268) and Syn11(SEQ ID NO:273). The full-length nucleic acid molecule encoding thepermutein protein was created and amplified from annealed fragments “NewN-termini fragment” and “New C-termini fragment” using oligonucleotideprimers Syn10 (SEQ ID NO:272) and Syn11 (SEQ ID NO:273).

[0247] The resulting amplified nucleic acid molecule was digested withrestriction endonucleases NcoI and EcoRI, and purified using theQIAquick PCR purification kit (Qiagen, Valencia, Calif.). PlasmidpMON33719 was digested with restriction endonucleases NcoI and EcoRI,and gel purified, resulting in an approximately 3900 base pair vectorfragment. The purified restriction fragments were combined and ligatedusing T4 DNA ligase.

[0248] The ligation reaction mixture was used to transform E. colistrain DH5α cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on ampicillin-containing plates. Plasmid DNA wasisolated and sequenced to confirm the presence of the correct insert.The resulting plasmid was designated pMON40704. Plasmid pMON40704 wasdigested with restriction endonuclease NotI, and the resulting 2200 bpDNA fragment was purified using the QIAquick PCR purification kit(Qiagen, Valencia, Calif.). Plasmid pMON30460 was digested withrestriction endonuclease NotI, and gel purified, resulting in anapproximately 4200 base pair vector fragment. The purified restrictionfragments were combined and ligated using T4 DNA ligase.

[0249] The ligation reaction mixture was used to transform E. colistrain DH5αα cells (Life Technologies, Gaithersburg, Md.). Transformantbacteria were selected on plates containing kanamycin. The resultingplasmid was designated pMON40705 (containing SEQ ID NO:274, encodingprotein sequence SEQ ID NO:275). Plasmid pMON40705 encodes a permuteinprotein with a “breakpoint” at positions 268/269 of the wild typepatatin protein sequence (SEQ ID NO:2). The first 23 amino acids of SEQID NO:2 are a signal peptide sequence which is cleaved in the matureprotein.

Example 18 Transient Expression of Protein in Corn Leaf Protoplasts

[0250] Plasmids pMON40701, pMON40703, and pMON40705 (all containing thenative signal sequence for vacuolar targeting) were separatelyelectroporated into corn leaf protoplasts as described by Sheen (PlantCell 3: 225-245, 1991). Protein was extracted with glass beads and thesupernatant was assayed for protein expression using ELISA for patatinand NPTII. Expression of protein by the transformed corn protoplasts wasconfirmed by Western blot analysis. Expression results are shown inTable 9. TABLE 9 ELISA data Normalized Expression Patatin ELISA NPTIIELISA (Patatin ELISA/ Sample (μg/mL) (μg/mL) NPTII ELISA) pMON40701 1.10.6 1.8 pMON40703 2.1 0.3 7.0 pMON40705 1.3 0.6 2.2

[0251] The results indicate that the permutein encoded by plasmidpMON40703 surprisingly shows approximately 4-fold higher expressioncompared to the wild type enzyme.

Example 19 Deglycosylation of Protein Sequences

[0252] This example provides evidence that glycosylation of cancontribute to the allergenicity of a protein. Accordingly, rationalsubstitution of amino acid residues likely to be the targets ofglycosylation within a subject allergen protein may reduce or eliminatethe allergenic properties of the protein without adversely affecting theenzymatic, insecticidal, antifungal or other functional properties ofthe protein.

[0253] Glycosylation commonly occurs as either N-linked or O-linkedforms. N-linked glycosylation usually occurs at the motifAsn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro (Kasturi, L. etal., Biochem J. 323: 415-519, 1997; Melquist, J. L. et al., Biochemistry37: 6833-6837, 1998). O-linked glycosylation occurs between the hydroxylgroup of serine or threonine and an amino sugar.

[0254] Site directed mutagenesis of selected asparagine, serine, orthreonine may be used to reduce or eliminate the glycosylation ofpatatin proteins. A search of SEQ ID NO:2 for the Asn-Xaa-Ser/Thr motifreveals one occurrence at amino acid positions 202-204. Mutagenizationof the nucleic acid sequence encoding this region results in a reducedallergenicity of the encoded protein.

[0255] In order to test this approach to reducing allergenicity ofpatatin proteins, two sets of experiments were performed: a) productionof patatin proteins in Escherichia coli, which do not glycosylateproteins; and b) production of patatin proteins with an N202Q sitedirected mutation.

[0256] Antibodies obtained from patients HS-07 and G15-MON (not potatoallergic) did not show specific binding to wild type patatin, patatinproduced in E. coli, or the N202Q variant. Antibodies obtained frompatient HS-01 (potato allergic) bound to wild type patatin, but not topatatin produced in E. coli or the N202Q variant. Antibodies obtainedfrom patient HS-02 (potato allergic) bound strongly to wild typepatatin, but extremely weakly to patatin produced in E. coli, andbinding to the N202Q variant resembled vector controls. Antibodiesobtained from patient HS-03 (potato allergic) bound to wild typepatatin, but not to patatin produced in E. coli or the N202Q variant.Antibodies obtained from patient HS-05 (potato allergic) bound to wildtype patatin, but very weakly to patatin produced in E. coli and theN202Q variant. Antibodies obtained from patient HS-06 (potato allergic)strongly bound wild type patatin, the N202Q variant, and to patatinproduced in E. coli. These results strongly suggest that glycosylationis at least partially responsible for the antigenic properties ofpatatin proteins, and that site directed mutagenesis may be used toreduce or eliminate specific antibody binding. Mutagenesis at position202 of SEQ ID NO:2 may be useful for reducing or eliminating specificantibody binding.

[0257] The deglycosylation approach was also tested using a patatinhomolog, Pat17. As demonstrated above, patatin epitopes exhibiting IgEbinding were identified, and each contained a Tyr residue. Substitutionof these Tyr residues within each epitope led to loss of IgE binding.Site-directed mutagenesis was used to produce variants with individualand multiple Tyr substitutions in the protein, which was expressed inPichia pastoris and assessed for enzyme activity. All the variants werefound to have enzymatic activity no less than the wild type protein. Asingle variant with all 5 tyrosine residues substituted withphenylalinine was found to have insecticidal activity no less than theunsubstituted protein and was expressed in E. coli to produce thenon-glycosylated version. The E. coli 5-“Tyr to Phe” variant wasassessed for IgE binding. An isozyme of patatin, designated Pat17, wasalso expressed in corn to produce a plant glycoprotein and in E. coli toproduce a nonglycosylated protein. Sera of seven patients (fiveexhibiting potato allergy and one exhibiting other allergies but noallergy to potatoes) were were used to assay Pat17 or Pat17 variantbinding by immunoblot assay. Four of the five sera from patientsexhibiting potato allergy showed either very weak or no binding to wildtype patatin expressed in E. coli but did bind to the 5-Tyr variant.Serum from one patient exhibiting potato allergy showed strong bindingto recombinant wild type patatin protein expressed in E. coli but weakbinding to the 5-Tyr variant. Sera from all five patients exhibitingpotato allergy bound strongly to patatin expressed in corn and nativepatatin present in potatoes. Serum from a control patient allergic toeggs, milk, peanuts and seafood, but exhibiting no allergy to potatoesshowed no binding to patatin expressed in E. coli but did bind topatatin expressed in corn. Immunoblot results suggested that the sugarmoiety in patatin is a non-specific IgE binding epitope and thepolypeptide portion of patatin also contains immunogenic IgE epitopes.

[0258] Patients who suffer from potato allergy were identified at JohnsHopkins Clinic (Baltimore, Md.) and were evaluated for potato allergyusing clinical criteria outlined in Table 2.

[0259] Serum was obtained from patients with convincing clinical historyof potato allergy. The convincing history was defined as being one ormore of the following: a) positive potato allergic reaction as evaluatedby double-blind placebo-control food challenge b) anaphylaix and/orhospitalization due to the consumption of potatoes or c) dramatic skintest results.

[0260] Peptide Synthesis

[0261] Peptides were synthesized on cellulose membranes using the SPOTSsystem (Genosys Biotechnologies, TX). Membranes were stored at −20° C.until use.

[0262] Site Directed Mutagenesis

[0263] Site specific mutations were introduced into patatin by firstincorporating part of the a-factor signal sequence (Pichia expressionmanual, Invitrogen, Carlsbad, Calif.) to the patatin gene using PCR.Primers used for the PCR wereGGAGCTCGAGAAAAGAGAGGCTGAAGCTCAGTTGGGAGAAATGGTGACTGTTCT (XhoI site initalics) and GGTCTAGAG GAATTCTCATTAATAAGAAG (EcoRI site in italics). Theprimers contained restriction sites to facilitate cloning into Pichiapastoris yeast secretion vector pPIC9 (GenBank accession number Z46233;Invitrogen, Carlsbad, Calif.). Typical PCR conditions are 25 cycles 94°C. denaturation for 1 minute, 45° C. annealing for one minute and 72° C.extension for 2 minutes; plus one cycle 72° C. extension for 10 minutes.A 50 mL reaction contained 30 pmol of each primer and 1 mg of templateDNA; and 1×PCR buffer with MgCl₂, 200 mM dGTP, 200 mM dATP, 200 mM dTTP,200 mM dCTP, 2.5 units of Pwo DNA polymerase. PCR reactions areperformed in RoboCycler Gradient 96 Temperature Cycler (Stratagene, LaJolla, Calif.).

[0264] The amplified patatin gene was digested with restriction enzymesXhoI and EcoRI and cloned into the pBluescript vector (Stratagene, LaJolla, Calif.), digested with the same two restriction enzymes. Thetemplate plasmid DNA used for the PCR was pMON26820. The resultingplasmid (pMON 26869) was used for oligonucleotide-directed mutagenesisusing the Bio-Rad mutagenesis kit based on the method of Kunkel et al.,Proc Natl Acad Sci USA 82, 477-92 (1985). Briefly, single-strandedpMON26869 was used as template for mutagenesis and was prepared bysuperinfection of plasmid containing cells with M13K07 (Gorman et al.,DNA and Protein Engineering techniques 2, 3-10 (1990)). DNA purifiedfrom transformed DH5a E. coli colonies was used for sequencedetermination. Sequencing was performed using the ABI PRISM sequencingkit (Perkin Elmer Biosystems, Foster City, Calif.).

[0265] Protein Expression in Pichia pastoris

[0266] Plasmids containing the mutations in the patatin gene weredigested with restriction enzymes XhoI and EcoRI. The patatin nucleicacid fragment was then ligated into the pPIC9 vector (Invitrogen,Carlsbad, Calif.), digested with the same two restriction enzymes toafford plasmid pMON37401. Pichia pastoris KM71 cells were electroporatedwith pMON37401 containing the appropriate mutation. The resultingtransformed cells were used to produce protein in Pichia pastoris usingthe procedure supplied by the manufacturer (Invitrogen, Carlsbad,Calif.). The proteins were purified in the same way as the proteinsexpressed in E. coli (see below).

[0267] Western Blotting of Proteins

[0268] Protein samples were electrophoresed by SDS-PAGE andelectroblotted onto PVDF membrane (Millipore, Bedford Mass.). Proteinblots were processed by standard Western blotting (immunoblotting)techniques and were incubated in potato allergic serum diluted 1:5 inPBS buffer for 1 hour. After washing the blots 3 times with PBS, theblots were incubated in biotinylated anti-IgE (Johns Hopkins Hospital,Baltimore Md.) for 1 hour, followed by a 30 minute incubation inHRP-linked avidin (Promega, New York, N.Y.). IgE-reactive protein bandswere visualized by using the ECL system (Amersham Pharmacia Biotech,NJ). As a control, one blot was incubated in anti-IgE only. His-taggedglyphosate oxidase and potato extracts was prepared and provided forthis study by Regulatory Sciences, Monsanto Company. The peptides wereevaluated using the same incubation procedures as described above.

[0269] Expression and Purification of Patatin in Corn

[0270] An isozyme of patatin, Pat17, was generated for expression incorn using a modified plant optimized gene sequence as described byBrown et al (U.S. Pat. No. 5,689,052). All the constructs contained thenative 23 amino acid signal peptide for vacuolar targeting. Corn wastransformed by microprojectile bombardment (Morrish et al., inTransgenic plants. Fundamentals and Applications (ed. Hiatt, A.) 133-171(Marcel Dekker, New York, 1993); Songstad et al., In Vitro Cell DevBiol—Plant 32, 179-183 (1996)). Protein from the transformed corn plantswas purified by first grinding the leaves in liquid nitrogen andextracting the protein using 25 mM Tris/HCl. The plant extract wasfurther dialyzed against 25 mM Tris/HCl pH 7.5. The plant extract wasthen loaded onto Mono Q HR 10/10 anion-exchange column (AmershamPharmacia, NJ) equilibrated with 25 mM Tris/HCl pH 7.5 (buffer A). Theprotein was eluted with 25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B) usinga linear gradient of 0-100% buffer B using an HPLC system (Shimadzu).Fractions containing protein were assayed for esterase activity anddialyzed against 25 mM Tris/HCl pH 7.5, 1 M Ammonium Sulfate (buffer C).The protein was purified to homogeneity by loading onto aphenyl-Sepharose 16/10 column (Amersham Pharmacia, NJ) equilibrated withbuffer C. Esterase active fractions were pooled and dialyzed against 25mM Tris/HCl pH 7.5.

[0271] Expression and Purification of Patatin in E. coli

[0272] Pat17 was expressed in E. coli using the pET expression system(Novagen, WI). The coding region of the mature Pat17 gene (without itssignal peptide) was amplified by PCR using the primers5′-GGGCCATGGCGCAGTTGGGAGAAATGGTG-3′ (NcoI site in italics) and5′-AACAAAGCTTCTTATTGAGGTGCGGCCGCTTGCATGC-3′ (NotI site in italics) usingstandard PCR reaction conditions as described in the Gene Amp kit(Perkin-Elmer Cetus, CT) and an annealing temperature of 40° C. Thetemplate was plasmid pMON26820. The resulting DNA was digested with NcoIand NotI and cloned into a modified pET24d plasmid, designed to add anN-terminal hexa-histidine tag to the protein. The correct sequence ofthe PCR product was verified by sequencing, and the plasmid wastransformed into E. coli BL21 (DE3), and transformants selected on LBcontaining 25 mg/mL kanamycin. The expression strain was grown in LBcontaining 25 mg/mL kanamycin and induced for 8 hrs at 28° C. with 1 mMIPTG. Cells were harvested and washed in 50 mM Tris/HCl pH 8.5, 150 mMNaCl, and lysed by French Press at 20,000 psi. His-tagged protein wasrecovered in the soluble fraction of lysed cells and subsequentlypurified using Ni-NTA resin as described in the QIAexpressionist manual(Qiagen CA). The partially purified protein was then dialyzed against 25mM Tris/HCl pH 7.5 (buffer A) and loaded onto Mono Q HR 10/10anion-exchange column (Amersham Pharmacia, NJ) equilibrated with bufferA. The protein was eluted with 25 mM Tris/HCl pH 7.5, 1 M KCl (buffer B)using a linear gradient of 0-100% buffer B run over 30 min at a flowrate of 4 mL/min using an HPLC system (Shimadzu). Fractions containingprotein were assayed for esterase activity. Esterase active fractionswere pooled, concentrated and dialyzed against 25 mM Tris/HCl pH 7.5 andstored at 4° C.

[0273] Enzyme Activity Assays

[0274] Enzyme activity was measured as described previously usingp-nitrophenyl caprate (Sigma, MO) as a substrate, dissolved indimethylsulfoxide (5 mM stock solution) and diluted in 4% Triton X-100,1% SDS to a final concentration of 1 mM. For the assay, 20 mL of proteinsolution was added to a mixture of 25 mL of the 1 mM substrate solutionand 80 mL of 50 mM Tris pH 8.5. The enzyme activity was monitored at 405nm in 6 sec interval for a period of 10 min. Esterase activity wasexpressed as DOD min⁻¹mg⁻¹ protein.

[0275] Insect Bioassay

[0276] The protein was also assayed for activity against larvae ofDiabrotica virigifera (Western corn rootworm) by overlaying the testsample on an agar diet similar to that described previously (Marrone etal., J. Econ. Entom. 78, 290-3 (1985)). Proteins to be tested werediluted in 25 mM Tris/HCl pH 7.5 and overlayed on the diet surface.Neonate larvae were allowed to feed on the diet and mortality and growthstunting were evaluated after 6 days.

[0277] IgE Binding Epitopes on Patatin

[0278] A panel of eighty-nine overlapping peptides representing theamino acid sequence of patatin were synthesized to determine the regionsresponsible for IgE binding. Each peptide was 10 amino acids long andconsisted of 6 amino acid overlap between the consecutive peptides. Thepeptides were evaluated for IgE binding with five different potatoallergic patient sera. Patatin has 3 major epitopes. These major IgEbinding regions represent amino acids 184-193, 188-197, 269-278 and360-369. Other minor IgE binding regions represent amino acids 104-113,138-147 and 316-325. The amino acids essential for IgE binding in eachmajor and minor epitopes were determined by synthesizing peptides withsingle amino acid changes at each position by individually substitutingan alanine residue at each non-alanine position in the epitopes. Theresulting alanine substituted peptides were evaluated for IgE binding.Result effective substitutions were identified by a reduction in IgEbinding with respect to the non-substituted peptide sequence. It wasvery interesting to note that all the epitopes contained a Tyr residueand substitution of this Tyr for Ala or Phe eliminated IgE binding.

[0279] Enzyme and Bioactivity

[0280] The Tyr residues identified to be critical for IgE binding ineach of the epitopes were substituted with Phe either individually or inconcert using site-directed mutagenesis. All the variants were expressedin Pichia pastoris and assessed for enzyme activity and insecticidalactivity. The variants included Y106F, Y129F, Y185F, Y193F, Y270F,Y316F, Y362F, Y185F/Y193F, Y185F/Y193F/Y270F/Y316F/Y362F (5-Tyr) andY106F/Y129F/Y185F/Y193F/Y270F/Y316F/Y362F (7-Tyr). All the variantsmaintained enzyme activity. The 5-Tyr and 7-Tyr variants were thenassessed for insecticidal activity by overlaying protein (200 ppm finalconcentration). The proteins caused significant stunting of the larvalgrowth as measured by the weight of the larvae after 6 days with the5-Tyr variant showing higher insecticidal activity compared to the 7-Tyrand wild type proteins. The 7-Tyr variant was unstable upon long termstorage at 4° C. and thus was not pursued further.

[0281] Immunoblotting

[0282] In order to test if the glycan moiety on patatin was importantfor binding of IgE, Pat17 was expressed in E. coli to produce anonglycosylated protein and in corn to produce a plant glycosylatedprotein. The 5-Tyr variant was also expressed in E. coli to assess theindividual contribution of the linear epitopes without the glycan moietyon the protein. The proteins were tested for binding to IgE using serafrom five patients with allergy to potatoes and sera from one patientwith allergies to many things but no allergy to potatoes. Proteins fromboth corn and E. coli were purified to homogeneity. These proteins weretransferred to PVDF membrane (Millipore, MA) and subsequently probedwith sera from patients with and without allergy to potatoes. AHis-tagged glyphosate oxidase control was included in all the studies toverify that the His-tag did not affect the binding of IgE. Serumobtained from patient HS-07 (no allergy to potatoes) did not bind Pat17expressed in E. coli but showed good binding to Pat17 from corn and alsoa protein at the same molecular weight in potato extract. It isinteresting to note that this sera also showed strong binding to anotherprotein (>46 kDa) in the potato. Sera from patients HS-01, HS-02, HS-03,HS-05 (allergy to potatoes) shows strong binding to Pat17 expressed incorn, but very weak to no binding to Pat17 produced in E. coli. Also,the sera from patients HS-01, HS-2, HS-03 and HS-05 bound to a proteinof similar molecular weight in the potato extract. Sera from patientsHS-01, HS-02 and HS-03 also showed binding to another protein in potatoextract of a lower molecular weight (<30 kDa). Serum obtained frompatient HS-06 (allergic to potatoes) showed very strong binding to wildtype patatin expressed in both corn and E. coli but weaker binding tothe 5-Tyr variant expressed in E. coli. Sera from HS-06 also showed verystrong binding to a protein in potato extract with similar molecularweight as patatin. The sera from all the patients showed no binding toHis-tagged glyphosate oxidase indicating that the His-tag does not bindIgE. These results strongly suggest that the glycan moiety on Pat17 isresponsible for IgE binding in some potato allergic patients and linearepitopes also contribute to the antigenicity of patatin.

Example 20 Alternative Nucleic Acid and Protein Sequences

[0283] For future variations of the patatin protein, sequences showinghigh similarity to the sequences disclosed herein could be used inproducing deallergenized patatin proteins and permuteins. For example, aBLAST search (Altschul, S. F. et al., J. Mol. Biol. 215: 403-410, 1990)can be performed to identify additional patatin sequences. Sources otherthan those disclosed herein can be used to obtain a patatin nucleic acidsequence, and the encoded patatin protein. Furthermore, subunitsequences from different organisms can be combined to create a novelpatatin sequence incorporating structural, regulatory, and enzymaticproperties from different sources.

Example 21 Nucleic Acid Mutation and Hybridization

[0284] Variations in the nucleic acid sequence encoding a patatinprotein may lead to mutant patatin protein sequences that displayequivalent or superior enzymatic characteristics when compared to thesequences disclosed herein. This invention accordingly encompassesnucleic acid sequences which are similar to the sequences disclosedherein, protein sequences which are similar to the sequences disclosedherein, and the nucleic acid sequences that encode them. Mutations caninclude deletions, insertions, truncations, substitutions, fusions,shuffling of subunit sequences, and the like.

[0285] Mutations to a nucleic acid sequence can be introduced in eithera specific or random manner, both of which are well known to those ofskill in the art of molecular biology. A myriad of site-directedmutagenesis techniques exist, typically using oligonucleotides tointroduce mutations at specific locations in a nucleic acid sequence.Examples include single strand rescue (Kunkel, T. Proc. Natl. Acad. Sci.U.S.A., 82: 488-492, 1985), unique site elimination (Deng and Nickloff,Anal. Biochem. 200: 81, 1992), nick protection (Vandeyar, et al. Gene65: 129-133, 1988), and PCR (Costa, et al. Methods Mol. Biol. 57: 31-44,1996). Random or non-specific mutations can be generated by chemicalagents (for a general review, see Singer and Kusmierek, Ann. Rev.Biochem. 52: 655-693, 1982) such as nitrosoguanidine (Cerda-Olmedo etal., J. Mol. Biol. 33: 705-719, 1968; Guerola, et al. Nature New Biol.230: 122-125, 1971) and 2-aminopurine (Rogan and Bessman, J. Bacteriol.103: 622-633, 1970), or by biological methods such as passage throughmutator strains (Greener et al. Mol. Biotechnol. 7: 189-195, 1997).

[0286] Nucleic acid hybridization is a technique well known to those ofskill in the art of DNA manipulation. The hybridization properties of agiven pair of nucleic acids is an indication of their similarity oridentity. Mutated nucleic acid sequences can be selected for theirsimilarity to the disclosed patatin nucleic acid sequences on the basisof their hybridization to the disclosed sequences. Low stringencyconditions can be used to select sequences with multiple mutations. Onemay wish to employ conditions such as about 0.15 M to about 0.9 M sodiumchloride, at temperatures ranging from about 20° C. to about 55° C. Highstringency conditions can be used to select for nucleic acid sequenceswith higher degrees of identity to the disclosed sequences. Conditionsemployed may include about 0.02 M to about 0.15 M sodium chloride, about0.5% to about 5% casein, about 0.02% SDS and/or about 0.1%N-laurylsarcosine, about 0.001 M to about 0.03 M sodium citrate, attemperatures between about 50° C. and about 70° C. More preferably, highstringency conditions are 0.02 M sodium chloride, 0.5% casein, 0.02%SDS, 0.001 M sodium citrate, at a temperature of 50° C.

Example 22 Determination of Homologous and Degenerate Nucleic AcidSequences

[0287] Modification and changes can be made in the sequence of theproteins of the present invention and the nucleic acid segments whichencode them and still obtain a functional molecule that encodes aprotein with desirable properties. The following is a discussion basedupon changing the amino acid sequence of a protein to create anequivalent, or possibly an improved, second-generation molecule. Theamino acid changes can be achieved by changing the codons of the nucleicacid sequence, according to the codons given in Table 10. TABLE 10 Codondegeneracies of amino acids One Amino acid letter Three letter CodonsAlanine A Ala GCA GCC GCG GCT Cysteine C Cys TGC TGT Aspartic acid D AspGAC GAT Glutamic acid E Glu GAA GAG Phenylalanine F Phe TTC TTT GlycineG Gly GGA GGC GGG GGT Histidine H His CAC CAT Isoleucine I Ile ATA ATCATT Lysine K Lys AAA AAG Leucine L Leu TTA TTG CTA CTC CTG CTTMethionine M Met ATG Asparagine N Asn AAC AAT Proline P Pro CCA CCC CCGCCT Glutamine Q Gln CAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGTSerine S Ser AGC AGT TCA TCC TCG TCT Threonine T Thr ACA ACC ACG ACTValine V Val GTA GTC GTG GTT Tryptophan W Trp TGG Tyrosine Y Tyr TAC TAT

[0288] Certain amino acids can be substituted for other amino acids in aprotein sequence without appreciable loss of enzymatic activity. It isthus contemplated that various changes can be made in the peptidesequences of the disclosed protein sequences, or their correspondingnucleic acid sequences without appreciable loss of the biologicalactivity.

[0289] In making such changes, the hydropathic index of amino acids canbe considered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, J. Mol. Biol., 157: 105-132,1982). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

[0290] Each amino acid has been assigned a hydropathic index on thebasis of their hydrophobicity and charge characteristics. These are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2);glutamate/glutamine/aspartate/asparagine (−3.5); lysine (−3.9); andarginine (−4.5).

[0291] It is known in the art that certain amino acids can besubstituted by other amino acids having a similar hydropathic index orscore and still result in a protein with similar biological activity,i.e., still obtain a biologically functional protein. In making suchchanges, the substitution of amino acids whose hydropathic indices arewithin ±2 is preferred, those within ±1 are more preferred, and thosewithin ±0.5 are most preferred.

[0292] It is also understood in the art that the substitution of likeamino acids can be made effectively on the basis of hydrophilicity. U.S.Pat. No. 4,554,101 (Hopp, T. P., issued Nov. 19, 1985) states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. The following hydrophilicity values have beenassigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate(+3.0±1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine/histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine/isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); and tryptophan (−3.4).

[0293] It is understood that an amino acid can be substituted by anotheramino acid having a similar hydrophilicity score and still result in aprotein with similar biological activity, i.e., still obtain abiologically functional protein. In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are more preferred, and those within ±0.5 aremost preferred.

[0294] As outlined above, amino acid substitutions are therefore basedon the relative similarity of the amino acid side-chain substituents,for example, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine. Changes which are not expected to be advantageous may alsobe used if these resulted in functional patatin proteins.

Example 23 Production of Patatin Proteins and Permuteins in Plants

[0295] Plant Vectors

[0296] In plants, transformation vectors capable of introducing nucleicacid sequences encoding patatin proteins and permuteins are easilydesigned, and generally contain one or more nucleic acid codingsequences of interest under the transcriptional control of 5′ and 3′regulatory sequences. Such vectors generally comprise, operativelylinked in sequence in the 5′ to 3′ direction, a promoter sequence thatdirects the transcription of a downstream heterologous structuralnucleic acid sequence in a plant; optionally, a 5′ non-translated leadersequence; a nucleic acid sequence that encodes a protein of interest;and a 3′ non-translated region that encodes a polyadenylation signalwhich functions in plant cells to cause the termination of transcriptionand the addition of polyadenylate nucleotides to the 3′ end of the mRNAencoding the protein. Plant transformation vectors also generallycontain a selectable marker. Typical 5′-3′ regulatory sequences includea transcription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal. Vectors for plant transformation have beenreviewed in Rodriguez et al. (Vectors: A Survey of Molecular CloningVectors and Their Uses, Butterworths, Boston., 1988), Glick et al.(Methods in Plant Molecular Biology and Biotechnology, CRC Press, BocaRaton, Fla., 1993), and Croy (Plant Molecular Biology Labfax, Hames andRickwood (Eds.), BIOS Scientific Publishers Limited, Oxford, UK., 1993).

[0297] Plant Promoters

[0298] Plant promoter sequences can be constitutive or inducible,environmentally- or developmentally-regulated, or cell- ortissue-specific. Often-used constitutive promoters include the CaMV 35Spromoter (Odell, J. T. et al., Nature 313: 810-812, 1985), the enhancedCaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins etal., Nucleic Acids Res. 20: 8451-8466, 1987), the mannopine synthase(mas) promoter, the nopaline synthase (nos) promoter, and the octopinesynthase (ocs) promoter. Useful inducible promoters include promotersinduced by salicylic acid or polyacrylic acids (PR-1, Williams, S. W. etal, Biotechnology 10: 540-543, 1992), induced by application of safeners(substituted benzenesulfonamide herbicides, Hershey, H. P. and Stoner,T. D., Plant Mol. Biol. 17: 679-690, 1991), heat-shock promoters (Ou-Leeet al., Proc. Natl. Acad. Sci. USA. 83: 6815-6819, 1986; Ainley et al.,Plant Mol. Biol. 14: 949-967, 1990), a nitrate-inducible promoterderived from the spinach nitrite reductase gene (Back et al., Plant Mol.Biol. 17: 9-18, 1991), hormone-inducible promoters (Yamaguchi-Shinozaki,K. et al., Plant Mol. Biol. 15: 905-912, 1990; Kares et al., Plant Mol.Biol. 15: 225-236, 1990), and light-inducible promoters associated withthe small subunit of RuBP carboxylase and LHCP gene families (Kuhlemeieret al., Plant Cell 1: 471, 1989; Feinbaum, R. L. et al., Mol. Gen.Genet. 226: 449-456, 1991; Weisshaar, B. et al., EMBO J. 10: 1777-1786,1991; Lam, E. and Chua, N. H., J. Biol. Chem. 266: 17131-17135, 1990;Castresana, C. et al., EMBO J. 7: 1929-1936, 1988; Schulze-Lefert etal., EMBO J. 8: 651, 1989). Examples of useful tissue-specific,developmentally-regulated promoters include the β-conglycinin 7Spromoter (Doyle, J. J. et al., J. Biol. Chem. 261: 9228-9238, 1986;Slighton and Beachy, Planta 172: 356-363, 1987), and seed-specificpromoters (Knutzon, D. S. et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624-2628, 1992; Bustos, M. M. et al., EMBO J. 10: 1469-1479, 1991; Lamand Chua, Science 248: 471, 1991; Stayton et al., Aust. J Plant.Physiol. 18: 507, 1991). Plant functional promoters useful forpreferential expression in seed plastids include those from plantstorage protein genes and from genes involved in fatty acid biosynthesisin oilseeds. Examples of such promoters include the 5′ regulatoryregions from such genes as napin (Kridl et al., Seed Sci. Res. 1:209-219, 1991), phaseolin, zein, soybean trypsin inhibitor, ACP,stearoyl-ACP desaturase, and oleosin. Seed-specific gene regulation isdiscussed in EP 0 255 378. Promoter hybrids can also be constructed toenhance transcriptional activity (Comai, L. and Moran, P. M., U.S. Pat.No. 5,106,739, issued Apr. 21, 1992), or to combine desiredtranscriptional activity and tissue specificity.

[0299] Plant Transformation and Regeneration

[0300] A variety of different methods can be employed to introduce suchvectors into plant protoplasts, cells, callus tissue, leaf discs,meristems, etcetera, to generate transgenic plants, includingAgrobacterium-mediated transformation, particle gun delivery,microinjection, electroporation, polyethylene glycol mediated protoplasttransformation, liposome-mediated transformation, etcetera (reviewed inPotrykus, I. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205-225,1991). In general, transgenic plants comprising cells containing andexpressing DNAs encoding patatin proteins and permuteins can be producedby transforming plant cells with a DNA construct as described above viaany of the foregoing methods; selecting plant cells that have beentransformed on a selective medium; regenerating plant cells that havebeen transformed to produce differentiated plants; and selecting atransformed plant which expresses the protein-encoding nucleotidesequence.

[0301] Specific methods for transforming a wide variety of dicots andobtaining transgenic plants are well documented in the literature(Gasser and Fraley, Science 244: 1293-1299, 1989; Fisk and Dandekar,Scientia Horticulturae 55: 5-36, 1993; Christou, Agro Food Industry HiTech, p. 17, 1994; and the references cited therein).

[0302] Successful transformation and plant regeneration have beenreported in the monocots as follows: asparagus (Asparagus officinalis;Bytebier et al., Proc. Natl. Acad. Sci. U.S.A. 84: 5345-5349, 1987);barley (Hordeum vulgarae; Wan and Lemaux, Plant Physiol. 104: 37-48,1994); maize (Zea mays; Rhodes, C. A. et al., Science 240: 204-207,1988; Gordon-Kamm et al., Plant Cell 2: 603-618, 1990; Fromm, M. E. etal., Bio/Technology 8: 833-839, 1990; Koziel et al., Bio/Technology 11:194-200, 1993); oats (Avena sativa; Somers et al., Bio/Technology 10:1589-1594, 1992); orchardgrass (Dactylis glomerata; Horn et al., PlantCell Rep. 7: 469-472, 1988); rice (Oryza sativa, including indica andjaponica varieties; Toriyama et al., Bio/Technology 6: 10, 1988; Zhanget al., Plant Cell Rep. 7: 379-384, 1988; Luo and Wu, Plant Mol. Biol.Rep. 6: 165-174, 1988; Zhang and Wu, Theor. Appl. Genet. 76: 835-840,1988; Christou et al., Bio/Technology 9: 957-962, 1991); rye (Secalecereale; De la Pena et al., Nature 325: 274-276, 1987); sorghum (Sorghumbicolor; Casas, A. M. et al., Proc. Natl. Acad. Sci. U.S.A. 90:11212-11216, 1993); sugar cane (Saccharum spp.; Bower and Birch, PlantJ. 2: 409-416, 1992); tall fescue (Festuca arundinacea; Wang, Z. Y. etal., Bio/Technology 10: 691-696, 1992); turfgrass (Agrostis palustris;Zhong et al., Plant Cell Rep. 13: 1-6, 1993); wheat (Triticum aestivum;Vasil et al., Bio/Technology 10: 667-674, 1992; Weeks, T. et al., PlantPhysiol. 102: 1077-1084, 1993; Becker et al., Plant J. 5: 299-307,1994), and alfalfa (Masoud, S. A. et al., Transgen. Res. 5: 313, 1996);Brassica (canola/oilseed rape) (Fry, J. Plant Cell Rep. 6: 321-325,1987); and soybean (Hinchee, M. Bio/Technol. 6: 915-922, 1988).

[0303] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations can be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related can be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

1 295 1 1158 DNA Solanum tuberosum 1 atggcaacta ctaaatcttt tttaattttaatatttatga tattagcaac tactagttca 60 acatttgctc agttgggaga aatggtgactgttcttagta ttgatggagg tggaattaga 120 gggatcattc cggctaccat tctcgaatttcttgaaggac aacttcagga aatggacaat 180 aatgcagatg caagacttgc agattactttgatgtaattg gaggaacaag tacaggaggt 240 ttattgactg ctatgataag tactccaaatgaaaacaatc gaccctttgc tgctgccaaa 300 gaaattgtac ctttttactt cgaacatggccctcagattt ttaatcctag tggtcaaatt 360 ttaggcccaa aatatgatgg aaaatatcttatgcaagttc ttcaagaaaa acttggagaa 420 actcgtgtgc atcaagcttt gacagaagttgtcatctcaa gctttgacat caaaacaaat 480 aagccagtaa tattcactaa gtcaaatttagcaaactctc cagaattgga tgctaagatg 540 tatgacataa gttattccac agcagcagctccaacatatt ttcctccgca ttactttgtt 600 actaatacta gtaatggaga tgaatatgagttcaatcttg ttgatggtgc tgttgctact 660 gttgctgatc cggcgttatt atccattagcgttgcaacga gacttgcaca aaaggatcca 720 gcatttgctt caattaggtc attgaattacaaaaaaatgc tgttgctctc attaggcact 780 ggcactactt cagagtttga taaaacatatacagcaaaag aggcagctac ctggactgct 840 gtacattgga tgttagttat acagaaaatgactgatgcag caagttctta catgactgat 900 tattaccttt ctactgcttt tcaagctcttgattcaaaaa acaattacct cagggttcaa 960 gaaaatgcat taacaggcac aactactgaaatggatgatg cttctgaggc taatatggaa 1020 ttattagtac aagttggtga aaacttattgaagaaaccag tttccgaaga caatcctgaa 1080 acctatgagg aagctctaaa gaggtttgcaaaattgctct ctgataggaa gaaactccga 1140 gcaaacaaag cttcttat 1158 2 386 PRTSolanum tuberosum 2 Met Ala Thr Thr Lys Ser Phe Leu Ile Leu Ile Phe MetIle Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr Phe Ala Gln Leu Gly Glu MetVal Thr Val Leu 20 25 30 Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile ProAla Thr Ile Leu 35 40 45 Glu Phe Leu Glu Gly Gln Leu Gln Glu Met Asp AsnAsn Ala Asp Ala 50 55 60 Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly ThrSer Thr Gly Gly 65 70 75 80 Leu Leu Thr Ala Met Ile Ser Thr Pro Asn GluAsn Asn Arg Pro Phe 85 90 95 Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr PheGlu His Gly Pro Gln 100 105 110 Ile Phe Asn Pro Ser Gly Gln Ile Leu GlyPro Lys Tyr Asp Gly Lys 115 120 125 Tyr Leu Met Gln Val Leu Gln Glu LysLeu Gly Glu Thr Arg Val His 130 135 140 Gln Ala Leu Thr Glu Val Val IleSer Ser Phe Asp Ile Lys Thr Asn 145 150 155 160 Lys Pro Val Ile Phe ThrLys Ser Asn Leu Ala Asn Ser Pro Glu Leu 165 170 175 Asp Ala Lys Met TyrAsp Ile Ser Tyr Ser Thr Ala Ala Ala Pro Thr 180 185 190 Tyr Phe Pro ProHis Tyr Phe Val Thr Asn Thr Ser Asn Gly Asp Glu 195 200 205 Tyr Glu PheAsn Leu Val Asp Gly Ala Val Ala Thr Val Ala Asp Pro 210 215 220 Ala LeuLeu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys Asp Pro 225 230 235 240Ala Phe Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu 245 250255 Ser Leu Gly Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260265 270 Lys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met Leu Val Ile Gln275 280 285 Lys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr LeuSer 290 295 300 Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu ArgVal Gln 305 310 315 320 Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met AspAsp Ala Ser Glu 325 330 335 Ala Asn Met Glu Leu Leu Val Gln Val Gly GluAsn Leu Leu Lys Lys 340 345 350 Pro Val Ser Glu Asp Asn Pro Glu Thr TyrGlu Glu Ala Leu Lys Arg 355 360 365 Phe Ala Lys Leu Leu Ser Asp Arg LysLys Leu Arg Ala Asn Lys Ala 370 375 380 Ser Tyr 385 3 54 DNA ArtificialSynthetic construct 3 ggagctcgag aaaagagagg ctgaagctca gttgggagaaatggtgactg ttct 54 4 29 DNA Artificial Synthetic construct 4 ggtctagaggaattctcatt aataagaag 29 5 1138 DNA Artificial Synthetic construct 5ggagctcgag aaaagagagg ctgaagctca gttgggagaa atggtgactg ttcttagtat 60tgatggaggt ggaattagag ggatcattcc ggctaccatt ctcgaatttc ttgaaggaca 120acttcaggaa atggacaata atgcagatgc aagacttgca gattactttg atgtaattgg 180aggaacaagt acaggaggtt tattgactgc tatgataagt actccaaatg aaaacaatcg 240accctttgct gctgccaaag aaattgtacc tttttacttc gaacatggcc ctcagatttt 300taatcctagt ggtcaaattt taggcccaaa atatgatgga aaatatctta tgcaagttct 360tcaagaaaaa cttggagaaa ctcgtgtgca tcaagctttg acagaagttg tcatctcaag 420ctttgacatc aaaacaaata agccagtaat attcactaag tcaaatttag caaactctcc 480agaattggat gctaagatgt atgacataag ttattccaca gcagcagctc caacatattt 540tcctccgcat tactttgtta ctaatactag taatggagat gaatatgagt tcaatcttgt 600tgatggtgct gttgctactg ttgctgatcc ggcgttatta tccattagcg ttgcaacgag 660acttgcacaa aaggatccag catttgcttc aattaggtca ttgaattaca aaaaaatgct 720gttgctctca ttaggcactg gcactacttc agagtttgat aaaacatata cagcaaaaga 780ggcagctacc tggactgctg tacattggat gttagttata cagaaaatga ctgatgcagc 840aagttcttac atgactgatt attacctttc tactgctttt caagctcttg attcaaaaaa 900caattacctc agggttcaag aaaatgcatt aacaggcaca actactgaaa tggatgatgc 960ttctgaggct aatatggaat tattagtaca agttggtgaa aacttattga agaaaccagt 1020ttccgaagac aatcctgaaa cctatgagga agctctaaag aggtttgcaa aattgctctc 1080tgataggaag aaactccgag caaacaaagc ttcttattaa tgagaattcc tctagacc 1138 6452 PRT Artificial Synthetic polypeptide 6 Met Arg Phe Pro Ser Ile PheThr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro ValAsn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala ValIle Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu ProPhe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr IleAla Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Glu Lys ArgGlu Ala Glu Ala Gln Leu Gly Glu Met Val Thr 85 90 95 Val Leu Ser Ile AspGly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr 100 105 110 Ile Leu Glu PheLeu Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala 115 120 125 Asp Ala ArgLeu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr 130 135 140 Gly GlyLeu Leu Thr Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg 145 150 155 160Pro Phe Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly 165 170175 Pro Gln Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp 180185 190 Gly Lys Tyr Leu Met Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg195 200 205 Val His Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp IleLys 210 215 220 Thr Asn Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala AsnSer Pro 225 230 235 240 Glu Leu Asp Ala Lys Met Tyr Asp Ile Ser Tyr SerThr Ala Ala Ala 245 250 255 Pro Thr Tyr Phe Pro Pro His Tyr Phe Val ThrAsn Thr Ser Asn Gly 260 265 270 Asp Glu Tyr Glu Phe Asn Leu Val Asp GlyAla Val Ala Thr Val Ala 275 280 285 Asp Pro Ala Leu Leu Ser Ile Ser ValAla Thr Arg Leu Ala Gln Lys 290 295 300 Asp Pro Ala Phe Ala Ser Ile ArgSer Leu Asn Tyr Lys Lys Met Leu 305 310 315 320 Leu Leu Ser Leu Gly ThrGly Thr Thr Ser Glu Phe Asp Lys Thr Tyr 325 330 335 Thr Ala Lys Glu AlaAla Thr Trp Thr Ala Val His Trp Met Leu Val 340 345 350 Ile Gln Lys MetThr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr 355 360 365 Leu Ser ThrAla Phe Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg 370 375 380 Val GlnGlu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala 385 390 395 400Ser Glu Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu 405 410415 Lys Lys Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu 420425 430 Lys Arg Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn435 440 445 Lys Ala Ser Tyr 450 7 367 PRT Artificial Syntheticpolypeptide 7 Glu Ala Glu Ala Gln Leu Gly Glu Met Val Thr Val Leu SerIle Asp 1 5 10 15 Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile LeuGlu Phe Leu 20 25 30 Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala Asp AlaArg Leu Ala 35 40 45 Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly GlyLeu Leu Thr 50 55 60 Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro PheAla Ala Ala 65 70 75 80 Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly ProGln Ile Phe Asn 85 90 95 Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp GlyLys Tyr Leu Met 100 105 110 Gln Val Leu Gln Glu Lys Leu Gly Glu Thr ArgVal His Gln Ala Leu 115 120 125 Thr Glu Val Val Ile Ser Ser Phe Asp IleLys Thr Asn Lys Pro Val 130 135 140 Ile Phe Thr Lys Ser Asn Leu Ala AsnSer Pro Glu Leu Asp Ala Lys 145 150 155 160 Met Tyr Asp Ile Ser Tyr SerThr Ala Ala Ala Pro Thr Tyr Phe Pro 165 170 175 Pro His Tyr Phe Val ThrAsn Thr Ser Asn Gly Asp Glu Tyr Glu Phe 180 185 190 Asn Leu Val Asp GlyAla Val Ala Thr Val Ala Asp Pro Ala Leu Leu 195 200 205 Ser Ile Ser ValAla Thr Arg Leu Ala Gln Lys Asp Pro Ala Phe Ala 210 215 220 Ser Ile ArgSer Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly 225 230 235 240 ThrGly Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala 245 250 255Ala Thr Trp Thr Ala Val His Trp Met Leu Val Ile Gln Lys Met Thr 260 265270 Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe 275280 285 Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala290 295 300 Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu Ala AsnMet 305 310 315 320 Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys LysPro Val Ser 325 330 335 Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu LysArg Phe Ala Lys 340 345 350 Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala AsnLys Ala Ser Tyr 355 360 365 8 24 DNA Artificial Synthetic construct 8atgttcgaag aaaaaaggta caat 24 9 24 DNA Artificial Synthetic construct 9ttgcataaga aattttccat cata 24 10 24 DNA Artificial Synthetic construct10 tgctgtggaa aaacttatgt cata 24 11 24 DNA Artificial Syntheticconstruct 11 cggaggaaaa aatgttggag ctgc 24 12 51 DNA ArtificialSynthetic construct 12 atgcggagga aaaaatgttg gagctgctgc tgtggaaaaacttatgtcat a 51 13 24 DNA Artificial Synthetic construct 13 ttttgctgtaaatgttttat caaa 24 14 24 DNA Artificial Synthetic construct 14aaccctgagg aaattgtttt ttga 24 15 24 DNA Artificial Synthetic construct15 agcttcctca aaggtttcag gatt 24 16 10 PRT Artificial Syntheticpolypeptide 16 Gln Leu Gly Glu Met Val Thr Val Leu Ser 1 5 10 17 10 PRTArtificial Synthetic polypeptide 17 Met Val Thr Val Leu Ser Ile Asp GlyGly 1 5 10 18 10 PRT Artificial Synthetic polypeptide 18 Leu Ser Ile AspGly Gly Gly Ile Arg Gly 1 5 10 19 10 PRT Artificial Syntheticpolypeptide 19 Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala 1 5 10 20 10 PRTArtificial Synthetic polypeptide 20 Arg Gly Ile Ile Pro Ala Thr Ile LeuGlu 1 5 10 21 10 PRT Artificial Synthetic polypeptide 21 Pro Ala Thr IleLeu Glu Phe Leu Glu Gly 1 5 10 22 10 PRT Artificial Syntheticpolypeptide 22 Leu Glu Phe Leu Glu Gly Gln Leu Gln Glu 1 5 10 23 10 PRTArtificial Synthetic polypeptide 23 Glu Gly Gln Leu Gln Glu Met Asp AsnAsn 1 5 10 24 10 PRT Artificial Synthetic polypeptide 24 Gln Glu Met AspAsn Asn Ala Asp Ala Arg 1 5 10 25 10 PRT Artificial Syntheticpolypeptide 25 Asn Asn Ala Asp Ala Arg Leu Ala Asp Tyr 1 5 10 26 10 PRTArtificial Synthetic polypeptide 26 Ala Arg Leu Ala Asp Tyr Phe Asp ValIle 1 5 10 27 10 PRT Artificial Synthetic polypeptide 27 Asp Tyr Phe AspVal Ile Gly Gly Thr Ser 1 5 10 28 10 PRT Artificial Syntheticpolypeptide 28 Val Ile Gly Gly Thr Ser Thr Gly Gly Leu 1 5 10 29 10 PRTArtificial Synthetic polypeptide 29 Thr Ser Thr Gly Gly Leu Leu Thr AlaMet 1 5 10 30 10 PRT Artificial Synthetic polypeptide 30 Gly Leu Leu ThrAla Met Ile Ser Thr Pro 1 5 10 31 10 PRT Artificial Syntheticpolypeptide 31 Ala Met Ile Ser Thr Pro Asn Glu Asn Asn 1 5 10 32 10 PRTArtificial Synthetic polypeptide 32 Thr Pro Asn Glu Asn Asn Arg Pro PheAla 1 5 10 33 10 PRT Artificial Synthetic polypeptide 33 Asn Asn Arg ProPhe Ala Ala Ala Lys Glu 1 5 10 34 10 PRT Artificial Syntheticpolypeptide 34 Phe Ala Ala Ala Lys Glu Ile Val Pro Phe 1 5 10 35 10 PRTArtificial Synthetic polypeptide 35 Lys Glu Ile Val Pro Phe Tyr Phe GluHis 1 5 10 36 10 PRT Artificial Synthetic polypeptide 36 Pro Phe Tyr PheGlu His Gly Pro Gln Ile 1 5 10 37 10 PRT Artificial Syntheticpolypeptide 37 Glu His Gly Pro Gln Ile Phe Asn Pro Ser 1 5 10 38 10 PRTArtificial Synthetic polypeptide 38 Gln Ile Phe Asn Pro Ser Gly Gln IleLeu 1 5 10 39 10 PRT Artificial Synthetic polypeptide 39 Pro Ser Gly GlnIle Leu Gly Pro Lys Tyr 1 5 10 40 10 PRT Artificial Syntheticpolypeptide 40 Ile Leu Gly Pro Lys Tyr Asp Gly Lys Tyr 1 5 10 41 10 PRTArtificial Synthetic polypeptide 41 Lys Tyr Asp Gly Lys Tyr Leu Met GlnVal 1 5 10 42 10 PRT Artificial Synthetic polypeptide 42 Lys Tyr Leu MetGln Val Leu Gln Glu Lys 1 5 10 43 10 PRT Artificial Syntheticpolypeptide 43 Gln Val Leu Gln Glu Lys Leu Gly Glu Thr 1 5 10 44 10 PRTArtificial Synthetic polypeptide 44 Glu Lys Leu Gly Glu Thr Arg Val HisGln 1 5 10 45 10 PRT Artificial Synthetic polypeptide 45 Glu Thr Arg ValHis Gln Ala Leu Thr Glu 1 5 10 46 10 PRT Artificial Syntheticpolypeptide 46 His Gln Ala Leu Thr Glu Val Val Ile Ser 1 5 10 47 10 PRTArtificial Synthetic polypeptide 47 Thr Glu Val Val Ile Ser Ser Phe AspIle 1 5 10 48 10 PRT Artificial Synthetic polypeptide 48 Ile Ser Ser PheAsp Ile Lys Thr Asn Lys 1 5 10 49 10 PRT Artificial Syntheticpolypeptide 49 Asp Ile Lys Thr Asn Lys Pro Val Ile Phe 1 5 10 50 10 PRTArtificial Synthetic polypeptide 50 Asn Lys Pro Val Ile Phe Thr Lys SerAsn 1 5 10 51 10 PRT Artificial Synthetic polypeptide 51 Ile Phe Thr LysSer Asn Leu Ala Asn Ser 1 5 10 52 10 PRT Artificial Syntheticpolypeptide 52 Ser Asn Leu Ala Asn Ser Pro Glu Leu Asp 1 5 10 53 10 PRTArtificial Synthetic polypeptide 53 Asn Ser Pro Glu Leu Asp Ala Lys MetTyr 1 5 10 54 10 PRT Artificial Synthetic polypeptide 54 Leu Asp Ala LysMet Tyr Asp Ile Ser Tyr 1 5 10 55 10 PRT Artificial Syntheticpolypeptide 55 Met Tyr Asp Ile Ser Tyr Ser Thr Ala Ala 1 5 10 56 10 PRTArtificial Synthetic polypeptide 56 Ser Tyr Ser Thr Ala Ala Ala Pro ThrTyr 1 5 10 57 10 PRT Artificial Synthetic polypeptide 57 Ala Ala Ala ProThr Tyr Phe Pro Pro His 1 5 10 58 10 PRT Artificial Syntheticpolypeptide 58 Thr Tyr Phe Pro Pro His Tyr Phe Val Thr 1 5 10 59 10 PRTArtificial Synthetic polypeptide 59 Pro His Tyr Phe Val Thr Asn Thr SerAsn 1 5 10 60 10 PRT Artificial Synthetic polypeptide 60 Val Thr Asn ThrSer Asn Gly Asp Glu Tyr 1 5 10 61 10 PRT Artificial Syntheticpolypeptide 61 Ser Asn Gly Asp Glu Tyr Glu Phe Asn Leu 1 5 10 62 10 PRTArtificial Synthetic polypeptide 62 Glu Tyr Glu Phe Asn Leu Val Asp GlyAla 1 5 10 63 10 PRT Artificial Synthetic polypeptide 63 Asn Leu Val AspGly Ala Val Ala Thr Val 1 5 10 64 10 PRT Artificial Syntheticpolypeptide 64 Gly Ala Val Ala Thr Val Ala Asp Pro Ala 1 5 10 65 10 PRTArtificial Synthetic polypeptide 65 Thr Val Ala Asp Pro Ala Leu Leu SerIle 1 5 10 66 10 PRT Artificial Synthetic polypeptide 66 Pro Ala Leu LeuSer Ile Ser Val Ala Thr 1 5 10 67 10 PRT Artificial Syntheticpolypeptide 67 Ser Ile Ser Val Ala Thr Arg Leu Ala Gln 1 5 10 68 10 PRTArtificial Synthetic polypeptide 68 Ala Thr Arg Leu Ala Gln Lys Asp ProAla 1 5 10 69 10 PRT Artificial Synthetic polypeptide 69 Ala Gln Lys AspPro Ala Phe Ala Ser Ile 1 5 10 70 10 PRT Artificial Syntheticpolypeptide 70 Pro Ala Phe Ala Ser Ile Arg Ser Leu Asn 1 5 10 71 10 PRTArtificial Synthetic polypeptide 71 Ser Ile Arg Ser Leu Asn Tyr Lys LysMet 1 5 10 72 10 PRT Artificial Synthetic polypeptide 72 Leu Asn Tyr LysLys Met Leu Leu Leu Ser 1 5 10 73 10 PRT Artificial Syntheticpolypeptide 73 Lys Met Leu Leu Leu Ser Leu Gly Thr Gly 1 5 10 74 10 PRTArtificial Synthetic polypeptide 74 Leu Ser Leu Gly Thr Gly Thr Thr SerGlu 1 5 10 75 10 PRT Artificial Synthetic polypeptide 75 Thr Gly Thr ThrSer Glu Phe Asp Lys Thr 1 5 10 76 10 PRT Artificial Syntheticpolypeptide 76 Ser Glu Phe Asp Lys Thr Tyr Thr Ala Lys 1 5 10 77 10 PRTArtificial Synthetic polypeptide 77 Lys Thr Tyr Thr Ala Lys Glu Ala AlaThr 1 5 10 78 10 PRT Artificial Synthetic polypeptide 78 Ala Lys Glu AlaAla Thr Trp Thr Ala Val 1 5 10 79 10 PRT Artificial Syntheticpolypeptide 79 Ala Thr Trp Thr Ala Val His Trp Met Leu 1 5 10 80 10 PRTArtificial Synthetic polypeptide 80 Ala Val His Trp Met Leu Val Ile GlnLys 1 5 10 81 10 PRT Artificial Synthetic polypeptide 81 Met Leu Val IleGln Lys Met Thr Asp Ala 1 5 10 82 10 PRT Artificial Syntheticpolypeptide 82 Gln Lys Met Thr Asp Tyr Tyr Leu Ser Thr 1 5 10 83 10 PRTArtificial Synthetic polypeptide 83 Asp Ala Ala Ser Ser Tyr Met Thr AspTyr 1 5 10 84 10 PRT Artificial Synthetic polypeptide 84 Ser Tyr Met ThrAsp Tyr Tyr Leu Ser Thr 1 5 10 85 10 PRT Artificial Syntheticpolypeptide 85 Asp Tyr Tyr Leu Ser Thr Ala Phe Gln Ala 1 5 10 86 10 PRTArtificial Synthetic polypeptide 86 Ser Thr Ala Phe Gln Ala Leu Asp SerLys 1 5 10 87 10 PRT Artificial Synthetic polypeptide 87 Gln Ala Leu AspSer Lys Asn Asn Tyr Leu 1 5 10 88 10 PRT Artificial Syntheticpolypeptide 88 Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu 1 5 10 89 10 PRTArtificial Synthetic polypeptide 89 Tyr Leu Arg Val Gln Glu Asn Ala LeuThr 1 5 10 90 10 PRT Artificial Synthetic polypeptide 90 Gln Glu Asn AlaLeu Thr Gly Thr Thr Thr 1 5 10 91 10 PRT Artificial Syntheticpolypeptide 91 Leu Thr Gly Thr Thr Thr Glu Met Asp Asp 1 5 10 92 10 PRTArtificial Synthetic polypeptide 92 Thr Thr Glu Met Asp Asp Ala Ser GluAla 1 5 10 93 10 PRT Artificial Synthetic polypeptide 93 Asp Asp Ala SerGlu Ala Asn Met Glu Leu 1 5 10 94 10 PRT Artificial Syntheticpolypeptide 94 Glu Ala Asn Met Glu Leu Leu Val Gln Val 1 5 10 95 10 PRTArtificial Synthetic polypeptide 95 Glu Leu Leu Val Gln Val Gly Glu AsnLeu 1 5 10 96 10 PRT Artificial Synthetic polypeptide 96 Gln Val Gly GluAsn Leu Leu Lys Lys Pro 1 5 10 97 10 PRT Artificial Syntheticpolypeptide 97 Asn Leu Leu Lys Lys Pro Val Ser Glu Asp 1 5 10 98 10 PRTArtificial Synthetic polypeptide 98 Lys Pro Val Ser Glu Asp Asn Pro GluThr 1 5 10 99 10 PRT Artificial Synthetic polypeptide 99 Glu Asp Asn ProGlu Thr Tyr Glu Glu Ala 1 5 10 100 10 PRT Artificial Syntheticpolypeptide 100 Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe 1 5 10 101 10PRT Artificial Synthetic polypeptide 101 Glu Ala Leu Lys Arg Phe Ala LysLeu Leu 1 5 10 102 10 PRT Artificial Synthetic polypeptide 102 Arg PheAla Lys Leu Leu Ser Asp Arg Lys 1 5 10 103 10 PRT Artificial Syntheticpolypeptide 103 Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala 1 5 10 104 10PRT Artificial Synthetic polypeptide 104 Arg Lys Lys Leu Arg Ala Asn LysAla Ser 1 5 10 105 10 PRT Artificial Synthetic polypeptide 105 Asp TyrPhe Asp Val Ile Gly Gly Thr Ser 1 5 10 106 10 PRT Artificial Syntheticpolypeptide 106 Asp Tyr Phe Asp Val Ile Ala Gly Thr Ser 1 5 10 107 10PRT Artificial Synthetic polypeptide 107 Val Ile Gly Gly Thr Ser Thr GlyGly Leu 1 5 10 108 10 PRT Artificial Synthetic polypeptide 108 Val IleAla Gly Thr Ser Thr Gly Ala Leu 1 5 10 109 10 PRT Artificial Syntheticpolypeptide 109 Ala Phe Tyr Phe Glu His Gly Pro Gln Ile 1 5 10 110 10PRT Artificial Synthetic polypeptide 110 Pro Ala Tyr Phe Glu His Gly ProGln Ile 1 5 10 111 10 PRT Artificial Synthetic polypeptide 111 Pro PheAla Phe Glu His Gly Pro Gln Ile 1 5 10 112 10 PRT Artificial Syntheticpolypeptide 112 Pro Phe Tyr Ala Glu His Gly Pro Gln Ile 1 5 10 113 10PRT Artificial Synthetic polypeptide 113 Pro Phe Tyr Phe Ala His Gly ProGln Ile 1 5 10 114 10 PRT Artificial Synthetic polypeptide 114 Pro PheTyr Phe Glu Ala Gly Pro Gln Ile 1 5 10 115 10 PRT Artificial Syntheticpolypeptide 115 Pro Phe Tyr Phe Glu His Ala Pro Gln Ile 1 5 10 116 10PRT Artificial Synthetic polypeptide 116 Pro Phe Tyr Phe Glu His Gly AlaGln Ile 1 5 10 117 10 PRT Artificial Synthetic polypeptide 117 Pro PheTyr Phe Glu His Gly Pro Ala Ile 1 5 10 118 10 PRT Artificial Syntheticpolypeptide 118 Pro Phe Tyr Phe Glu His Gly Pro Gln Ala 1 5 10 119 10PRT Artificial Synthetic polypeptide 119 Thr Phe Tyr Leu Glu Asn Gly ProLys Ile 1 5 10 120 10 PRT Artificial Synthetic polypeptide 120 Pro PhePhe Phe Glu His Gly Pro Gln Ile 1 5 10 121 10 PRT Artificial Syntheticpolypeptide 121 Ala Tyr Leu Met Gln Val Leu Gln Glu Lys 1 5 10 122 10PRT Artificial Synthetic polypeptide 122 Lys Ala Leu Met Gln Val Leu GlnGlu Lys 1 5 10 123 10 PRT Artificial Synthetic polypeptide 123 Lys TyrAla Met Gln Val Leu Gln Glu Lys 1 5 10 124 10 PRT Artificial Syntheticpolypeptide 124 Lys Tyr Leu Ala Gln Val Leu Gln Glu Lys 1 5 10 125 10PRT Artificial Synthetic polypeptide 125 Lys Tyr Leu Met Ala Val Leu GlnGlu Lys 1 5 10 126 10 PRT Artificial Synthetic polypeptide 126 Lys TyrLeu Met Gln Ala Leu Gln Glu Lys 1 5 10 127 10 PRT Artificial Syntheticpolypeptide 127 Lys Tyr Leu Met Gln Val Ala Gln Glu Lys 1 5 10 128 10PRT Artificial Synthetic polypeptide 128 Lys Tyr Leu Met Gln Val Leu AlaGlu Lys 1 5 10 129 10 PRT Artificial Synthetic polypeptide 129 Lys TyrLeu Met Gln Val Leu Gln Ala Lys 1 5 10 130 10 PRT Artificial Syntheticpolypeptide 130 Lys Tyr Leu Met Gln Val Leu Gln Glu Ala 1 5 10 131 10PRT Artificial Synthetic polypeptide 131 Val Phe Leu His Asp Lys Ile LysSer Leu 1 5 10 132 10 PRT Artificial Synthetic polypeptide 132 Ala TyrSer Thr Ala Ala Ala Pro Thr Tyr 1 5 10 133 10 PRT Artificial Syntheticpolypeptide 133 Ser Ala Ser Thr Ala Ala Ala Pro Thr Tyr 1 5 10 134 10PRT Artificial Synthetic polypeptide 134 Ser Tyr Ala Thr Ala Ala Ala ProThr Tyr 1 5 10 135 10 PRT Artificial Synthetic polypeptide 135 Ser TyrSer Ala Ala Ala Ala Pro Thr Tyr 1 5 10 136 10 PRT Artificial Syntheticpolypeptide 136 Ser Tyr Ser Thr Ala Ala Ala Ala Thr Tyr 1 5 10 137 10PRT Artificial Synthetic polypeptide 137 Ser Tyr Ser Thr Ala Ala Ala ProAla Tyr 1 5 10 138 10 PRT Artificial Synthetic polypeptide 138 Ser TyrSer Thr Ala Ala Ala Pro Thr Ala 1 5 10 139 10 PRT Artificial Syntheticpolypeptide 139 Cys Ile Ser Thr Ser Ala Ala Pro Thr Tyr 1 5 10 140 10PRT Artificial Synthetic polypeptide 140 Ser Tyr Ser Thr Ala Ala Ala ProAla Phe 1 5 10 141 10 PRT Artificial Synthetic polypeptide 141 Ala PheAla Ala Ala Ala Ala Pro Thr Tyr 1 5 10 142 10 PRT Artificial Syntheticpolypeptide 142 Ser Tyr Ser Thr Ala Ala Ala Pro Thr Phe 1 5 10 143 10PRT Artificial Synthetic polypeptide 143 Ser Thr Ser Ala Ala Pro Thr TyrPhe Pro 1 5 10 144 10 PRT Artificial Synthetic polypeptide 144 Ser ThrSer Ala Ala Pro Thr Phe Phe Pro 1 5 10 145 10 PRT Artificial Syntheticpolypeptide 145 Ser Thr Ser Ala Ala Pro Thr Ala Phe Pro 1 5 10 146 10PRT Artificial Synthetic polypeptide 146 Ser Thr Ala Ala Ala Pro Thr PhePhe Pro 1 5 10 147 10 PRT Artificial Synthetic polypeptide 147 Ala AlaAla Ala Thr Tyr Phe Pro Pro His 1 5 10 148 10 PRT Artificial Syntheticpolypeptide 148 Ala Ala Ala Pro Ala Tyr Phe Pro Pro His 1 5 10 149 10PRT Artificial Synthetic polypeptide 149 Ala Ala Ala Pro Thr Ala Phe ProPro His 1 5 10 150 10 PRT Artificial Synthetic polypeptide 150 Ala AlaAla Pro Thr Tyr Ala Pro Pro His 1 5 10 151 10 PRT Artificial Syntheticpolypeptide 151 Ala Ala Ala Pro Thr Tyr Phe Ala Pro His 1 5 10 152 10PRT Artificial Synthetic polypeptide 152 Ala Ala Ala Pro Thr Tyr Phe ProAla His 1 5 10 153 10 PRT Artificial Synthetic polypeptide 153 Ala AlaAla Pro Thr Tyr Phe Pro Pro Ala 1 5 10 154 10 PRT Artificial Syntheticpolypeptide 154 Ser Ala Ala Pro Thr Tyr Phe Pro Ala His 1 5 10 155 10PRT Artificial Synthetic polypeptide 155 Ala Ala Ala Pro Ala Phe Phe ProPro His 1 5 10 156 10 PRT Artificial Synthetic polypeptide 156 Ala AlaAla Pro Pro Phe Phe Pro Pro His 1 5 10 157 10 PRT Artificial Syntheticpolypeptide 157 Ala Ala Ala Pro Thr Phe Phe Pro Pro His 1 5 10 158 10PRT Artificial Synthetic polypeptide 158 Ser Ile Ser Val Ala Thr Arg LeuAla Gln 1 5 10 159 10 PRT Artificial Synthetic polypeptide 159 Ala MetSer Met Leu Thr Lys Glu Val His 1 5 10 160 10 PRT Artificial Syntheticpolypeptide 160 Pro Ala Phe Ala Ser Ile Arg Ser Leu Asn 1 5 10 161 10PRT Artificial Synthetic polypeptide 161 Pro Asn Phe Asn Ala Gly Ser ProThr Glu 1 5 10 162 10 PRT Artificial Synthetic polypeptide 162 Lys MetLeu Leu Leu Ser Leu Gly Thr Gly 1 5 10 163 10 PRT Artificial Syntheticpolypeptide 163 Asn Tyr Leu Ile Ile Ser Val Gly Thr Gly 1 5 10 164 10PRT Artificial Synthetic polypeptide 164 Lys Met Leu Leu Leu Ser Leu GlyAla Gly 1 5 10 165 10 PRT Artificial Synthetic polypeptide 165 Ala GluPhe Asp Lys Thr Tyr Thr Ala Lys 1 5 10 166 10 PRT Artificial Syntheticpolypeptide 166 Ser Ala Phe Asp Lys Thr Tyr Thr Ala Lys 1 5 10 167 10PRT Artificial Synthetic polypeptide 167 Ser Glu Ala Asp Lys Thr Tyr ThrAla Lys 1 5 10 168 10 PRT Artificial Synthetic polypeptide 168 Ser GluPhe Ala Lys Thr Tyr Thr Ala Lys 1 5 10 169 10 PRT Artificial Syntheticpolypeptide 169 Ser Glu Phe Asp Ala Thr Tyr Thr Ala Lys 1 5 10 170 10PRT Artificial Synthetic polypeptide 170 Ser Glu Phe Asp Lys Ala Tyr ThrAla Lys 1 5 10 171 10 PRT Artificial Synthetic polypeptide 171 Ser GluPhe Asp Lys Thr Ala Thr Ala Lys 1 5 10 172 10 PRT Artificial Syntheticpolypeptide 172 Ser Glu Phe Asp Lys Thr Tyr Ala Ala Lys 1 5 10 173 10PRT Artificial Synthetic polypeptide 173 Ser Glu Phe Asp Lys Thr Tyr ThrAla Ala 1 5 10 174 10 PRT Artificial Synthetic polypeptide 174 Lys GlnAla Glu Lys Tyr Thr Ala Glu Gln 1 5 10 175 10 PRT Artificial Syntheticpolypeptide 175 Ser Glu Phe Asp Ala Ala Phe Ala Ala Ala 1 5 10 176 10PRT Artificial Synthetic polypeptide 176 Ser Glu Phe Asp Lys Thr Phe ThrAla Lys 1 5 10 177 10 PRT Artificial Synthetic polypeptide 177 Ala GluLys Tyr Thr Ala Glu Gln Cys Ala 1 5 10 178 10 PRT Artificial Syntheticpolypeptide 178 Ala Thr Tyr Thr Ala Lys Glu Ala Ala Thr 1 5 10 179 10PRT Artificial Synthetic polypeptide 179 Lys Ala Tyr Thr Ala Lys Glu AlaAla Thr 1 5 10 180 10 PRT Artificial Synthetic polypeptide 180 Lys ThrAla Thr Ala Lys Glu Ala Ala Thr 1 5 10 181 10 PRT Artificial Syntheticpolypeptide 181 Lys Thr Tyr Ala Ala Lys Glu Ala Ala Thr 1 5 10 182 10PRT Artificial Synthetic polypeptide 182 Lys Thr Tyr Thr Ala Ala Glu AlaAla Thr 1 5 10 183 10 PRT Artificial Synthetic polypeptide 183 Lys ThrTyr Thr Ala Lys Ala Ala Ala Thr 1 5 10 184 10 PRT Artificial Syntheticpolypeptide 184 Lys Thr Tyr Thr Ala Lys Glu Ala Ala Ala 1 5 10 185 10PRT Artificial Synthetic polypeptide 185 Glu Lys Tyr Thr Ala Glu Gln CysAla Lys 1 5 10 186 10 PRT Artificial Synthetic polypeptide 186 Ala AlaPhe Ala Ala Ala Glu Ala Ala Thr 1 5 10 187 10 PRT Artificial Syntheticpolypeptide 187 Lys Thr Phe Thr Ala Lys Glu Ala Ala Thr 1 5 10 188 10PRT Artificial Synthetic polypeptide 188 Gln Ala Leu His Cys Glu Lys LysTyr Leu 1 5 10 189 10 PRT Artificial Synthetic polypeptide 189 Gln AlaLeu Asp Ser Lys Ala Ala Tyr Leu 1 5 10 190 10 PRT Artificial Syntheticpolypeptide 190 Gln Ala Leu Asp Ser Lys Asn Asn Phe Leu 1 5 10 191 10PRT Artificial Synthetic polypeptide 191 Gln Ala Leu His Cys Glu Asn AsnPhe Leu 1 5 10 192 10 PRT Artificial Synthetic polypeptide 192 Cys GluLys Lys Tyr Leu Arg Ile Gln Asp 1 5 10 193 10 PRT Artificial Syntheticpolypeptide 193 Ser Lys Asn Asn Phe Leu Arg Val Gln Glu 1 5 10 194 10PRT Artificial Synthetic polypeptide 194 Ser Glu Asn Asn Tyr Leu Arg ValGln Glu 1 5 10 195 10 PRT Artificial Synthetic polypeptide 195 Ala LeuArg Val Gln Glu Asn Ala Leu Thr 1 5 10 196 10 PRT Artificial Syntheticpolypeptide 196 Tyr Ala Arg Val Gln Glu Asn Ala Leu Thr 1 5 10 197 10PRT Artificial Synthetic polypeptide 197 Tyr Leu Ala Val Gln Glu Asn AlaLeu Thr 1 5 10 198 10 PRT Artificial Synthetic polypeptide 198 Tyr LeuArg Ala Gln Glu Asn Ala Leu Thr 1 5 10 199 10 PRT Artificial Syntheticpolypeptide 199 Tyr Leu Arg Val Ala Glu Asn Ala Leu Thr 1 5 10 200 10PRT Artificial Synthetic polypeptide 200 Tyr Leu Arg Val Gln Ala Asn AlaLeu Thr 1 5 10 201 10 PRT Artificial Synthetic polypeptide 201 Tyr LeuArg Val Gln Glu Ala Ala Leu Thr 1 5 10 202 10 PRT Artificial Syntheticpolypeptide 202 Tyr Leu Arg Val Gln Glu Asn Ala Ala Thr 1 5 10 203 10PRT Artificial Synthetic polypeptide 203 Tyr Leu Arg Val Gln Glu Asn AlaLeu Ala 1 5 10 204 10 PRT Artificial Synthetic polypeptide 204 Tyr LeuArg Ile Gln Asp Asp Thr Leu Thr 1 5 10 205 10 PRT Artificial Syntheticpolypeptide 205 Tyr Leu Thr Val Ala Ala Ala Ala Leu Thr 1 5 10 206 10PRT Artificial Synthetic polypeptide 206 Phe Leu Arg Val Gln Glu Asn AlaLeu Thr 1 5 10 207 10 PRT Artificial Synthetic polypeptide 207 Asn AsnTyr Leu Arg Val Gln Glu Asn Ala 1 5 10 208 10 PRT Artificial Syntheticpolypeptide 208 Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr 1 5 10 209 10PRT Artificial Synthetic polypeptide 209 Asn Asn Phe Leu Arg Val Gln GluAsn Ala 1 5 10 210 10 PRT Artificial Synthetic polypeptide 210 Asn AlaTyr Leu Arg Val Gln Glu Asn Ala 1 5 10 211 10 PRT Artificial Syntheticpolypeptide 211 Ala Thr Tyr Glu Glu Ala Lys Leu Arg Phe 1 5 10 212 10PRT Artificial Synthetic polypeptide 212 Glu Ala Tyr Glu Glu Ala Leu LysArg Phe 1 5 10 213 10 PRT Artificial Synthetic polypeptide 213 Glu ThrAla Glu Glu Ala Leu Lys Arg Phe 1 5 10 214 10 PRT Artificial Syntheticpolypeptide 214 Glu Thr Tyr Ala Glu Ala Leu Lys Arg Phe 1 5 10 215 10PRT Artificial Synthetic polypeptide 215 Glu Thr Tyr Glu Ala Ala Leu LysArg Phe 1 5 10 216 10 PRT Artificial Synthetic polypeptide 216 Glu ThrTyr Glu Glu Ala Ala Lys Arg Phe 1 5 10 217 10 PRT Artificial Syntheticpolypeptide 217 Glu Thr Tyr Glu Glu Ala Leu Ala Arg Phe 1 5 10 218 10PRT Artificial Synthetic polypeptide 218 Glu Thr Tyr Glu Glu Ala Leu LysAla Phe 1 5 10 219 10 PRT Artificial Synthetic polypeptide 219 Glu ThrTyr Glu Glu Ala Leu Lys Arg Ala 1 5 10 220 10 PRT Artificial Syntheticpolypeptide 220 Gly Thr Asn Ala Gln Ser Leu Ala Asp Phe 1 5 10 221 10PRT Artificial Synthetic polypeptide 221 Glu Thr Tyr Glu Ala Ala Leu AlaAla Phe 1 5 10 222 10 PRT Artificial Synthetic polypeptide 222 Glu ThrPhe Glu Glu Ala Leu Lys Arg Phe 1 5 10 223 10 PRT Artificial Syntheticpolypeptide 223 Tyr Glu Glu Ala Leu Lys Thr Phe Ala Lys 1 5 10 224 10PRT Artificial Synthetic polypeptide 224 Phe Glu Glu Ala Leu Lys Arg PheAla Lys 1 5 10 225 10 PRT Artificial Synthetic polypeptide 225 Ala AlaLeu Lys Arg Phe Ala Lys Leu Leu 1 5 10 226 10 PRT Artificial Syntheticpolypeptide 226 Glu Ala Ala Lys Arg Phe Ala Lys Leu Leu 1 5 10 227 10PRT Artificial Synthetic polypeptide 227 Glu Ala Leu Ala Arg Phe Ala LysLeu Leu 1 5 10 228 10 PRT Artificial Synthetic polypeptide 228 Glu AlaLeu Lys Ala Phe Ala Lys Leu Leu 1 5 10 229 10 PRT Artificial Syntheticpolypeptide 229 Glu Ala Leu Lys Arg Ala Ala Lys Leu Leu 1 5 10 230 10PRT Artificial Synthetic polypeptide 230 Glu Ala Leu Lys Arg Phe Ala AlaLeu Leu 1 5 10 231 10 PRT Artificial Synthetic polypeptide 231 Glu AlaLeu Lys Arg Phe Ala Lys Ala Leu 1 5 10 232 10 PRT Artificial Syntheticpolypeptide 232 Glu Ala Leu Lys Arg Phe Ala Lys Leu Ala 1 5 10 233 10PRT Artificial Synthetic polypeptide 233 Gln Ser Leu Ala Asp Phe Ala LysGln Leu 1 5 10 234 10 PRT Artificial Synthetic polypeptide 234 Ala AlaLeu Ala Ala Phe Ala Lys Leu Leu 1 5 10 235 10 PRT Artificial Syntheticpolypeptide 235 Leu Ala Asp Phe Ala Lys Gln Leu Ser Asp 1 5 10 236 10PRT Artificial Synthetic polypeptide 236 Asp Phe Ala Lys Gln Leu Ser AspGlu Arg 1 5 10 237 10 PRT Artificial Synthetic polypeptide 237 Ala PheAla Ala Leu Leu Ser Asp Arg Lys 1 5 10 238 10 PRT Artificial Syntheticpolypeptide 238 Ser Thr Ala Ala Ala Pro Thr Tyr Phe Pro 1 5 10 239 10PRT Artificial Synthetic polypeptide 239 Leu Lys Arg Phe Ala Lys Leu LeuSer Asp 1 5 10 240 10 PRT Artificial Synthetic polypeptide 240 Gln AlaLeu Asp Ser Glu Asn Asn Phe Leu 1 5 10 241 10 PRT Artificial Syntheticpolypeptide 241 Ser Asp Leu Ala Asp Phe Ala Lys Gln Leu 1 5 10 242 55DNA Artificial Synthetic construct 242 ggagctcgag aaaagagagg ctgaagcttcattgaattac aaaaaaatgc tgttg 55 243 42 DNA Artificial Synthetic construct243 tcccaactgt cctggtccat aagaagcttt gtttgctcgg ag 42 244 36 DNAArtificial Synthetic construct 244 gcttcttatg gaccaggaca gttgggagaaatggtg 36 245 39 DNA Artificial Synthetic construct 245 ggtctagaggaattctcatt acctaattga agcaaatgc 39 246 1128 DNA Artificial Syntheticconstruct 246 tcgagaaaag agaggctgaa gcttcattga attacaaaaa aatgctgttgctctcattag 60 gcactggcac tacttcagag tttgataaaa catatacagc aaaagaggcagctacctgga 120 ctgctgtaca ttggatgtta gttatacaga aaatgactga tgcagcaagttcttacatga 180 ctgattatta cctttctact gcttttcaag ctcttgattc aaaaaacaattacctcaggg 240 ttcaagaaaa tgcattaaca ggcacaacta ctgaaatgga tgatgcttctgaggctaata 300 tggaattatt agtacaagtt ggtgaaaact tattgaagaa accagtttccgaagacaatc 360 ctgaaaccta tgaggaagct ctaaagaggt ttgcaaaatt gctctctgataggaagaaac 420 tccgagcaaa caaagcttct tatggaccag gacagttggg agaaatggtgactgttctta 480 gtattgatgg aggtggaatt agagggatca ttccggctac cattctcgaatttcttgaag 540 gacaacttca ggaaatggac aataatgcag atgcaagact tgcagattactttgatgtaa 600 ttggaggaac aagtacagga ggtttattga ctgctatgat aagtactccaaatgaaaaca 660 atcgaccctt tgctgctgcc aaagaaattg taccttttta cttcgaacatggccctcaga 720 tttttaatcc tagtggtcaa attttaggcc caaaatatga tggaaaatatcttatgcaag 780 ttcttcaaga aaaacttgga gaaactcgtg tgcatcaagc tttgacagaagttgtcatct 840 caagctttga catcaaaaca aataagccag taatattcac taagtcaaatttagcaaact 900 ctccagaatt ggatgctaag atgtatgaca taagttattc cacagcagcagctccaacat 960 attttcctcc gcattacttt gttactaata ctagtaatgg agatgaatatgagttcaatc 1020 ttgttgatgg tgctgttgct actgttgctg atccggcgtt attatccattagcgttgcaa 1080 cgagacttgc acaaaaggat ccagcatttg cttcaattag gtaatgag1128 247 366 PRT Artificial Synthetic polypeptide 247 Ser Leu Asn TyrLys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly Thr 1 5 10 15 Thr Ser GluPhe Asp Lys Thr Tyr Thr Ala Lys Glu Ala Ala Thr Trp 20 25 30 Thr Ala ValHis Trp Met Leu Val Ile Gln Lys Met Thr Asp Ala Ala 35 40 45 Ser Ser TyrMet Thr Asp Tyr Tyr Leu Ser Thr Ala Phe Gln Ala Leu 50 55 60 Asp Ser LysAsn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr Gly 65 70 75 80 Thr ThrThr Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu 85 90 95 Val GlnVal Gly Glu Asn Leu Leu Lys Lys Pro Val Ser Glu Asp Asn 100 105 110 ProGlu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser 115 120 125Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr Gly Pro Gly Gln 130 135140 Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly Ile Arg 145150 155 160 Gly Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu Gly Gln LeuGln 165 170 175 Glu Met Asp Asn Asn Ala Asp Ala Arg Leu Ala Asp Tyr PheAsp Val 180 185 190 Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala MetIle Ser Thr 195 200 205 Pro Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala LysGlu Ile Val Pro 210 215 220 Phe Tyr Phe Glu His Gly Pro Gln Ile Phe AsnPro Ser Gly Gln Ile 225 230 235 240 Leu Gly Pro Lys Tyr Asp Gly Lys TyrLeu Met Gln Val Leu Gln Glu 245 250 255 Lys Leu Gly Glu Thr Arg Val HisGln Ala Leu Thr Glu Val Val Ile 260 265 270 Ser Ser Phe Asp Ile Lys ThrAsn Lys Pro Val Ile Phe Thr Lys Ser 275 280 285 Asn Leu Ala Asn Ser ProGlu Leu Asp Ala Lys Met Tyr Asp Ile Ser 290 295 300 Tyr Ser Thr Ala AlaAla Pro Thr Tyr Phe Pro Pro His Tyr Phe Val 305 310 315 320 Thr Asn ThrSer Asn Gly Asp Glu Tyr Glu Phe Asn Leu Val Asp Gly 325 330 335 Ala ValAla Thr Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val Ala 340 345 350 ThrArg Leu Ala Gln Lys Asp Pro Ala Phe Ala Ser Ile Arg 355 360 365 248 55DNA Artificial Synthetic construct 248 ggagctcgag aaaagagagg ctgaagctaatactagtaat ggagatgaat atgag 55 249 39 DNA Artificial Synthetic construct249 ggtctagagg aattctcatt aagtaacaaa gtaatgcgg 39 250 1128 DNAArtificial Synthetic construct 250 tcgagaaaag agaggctgaa gctaatactagtaatggaga tgaatatgag ttcaatcttg 60 ttgatggtgc tgttgctact gttgctgatccggcgttatt atccattagc gttgcaacga 120 gacttgcaca aaaggatcca gcatttgcttcaattaggtc attgaattac aaaaaaatgc 180 tgttgctctc attaggcact ggcactacttcagagtttga taaaacatat acagcaaaag 240 aggcagctac ctggactgct gtacattggatgttagttat acagaaaatg actgatgcag 300 caagttctta catgactgat tattacctttctactgcttt tcaagctctt gattcaaaaa 360 acaattacct cagggttcaa gaaaatgcattaacaggcac aactactgaa atggatgatg 420 cttctgaggc taatatggaa ttattagtacaagttggtga aaacttattg aagaaaccag 480 tttccgaaga caatcctgaa acctatgaggaagctctaaa gaggtttgca aaattgctct 540 ctgataggaa gaaactccga gcaaacaaagcttcttatgg accaggacag ttgggagaaa 600 tggtgactgt tcttagtatt gatggaggtggaattagagg gatcattccg gctaccattc 660 tcgaatttct tgaaggacaa cttcaggaaatggacaataa tgcagatgca agacttgcag 720 attactttga tgtaattgga ggaacaagtacaggaggttt attgactgct atgataagta 780 ctccaaatga aaacaatcga ccctttgctgctgccaaaga aattgtacct ttttacttcg 840 aacatggccc tcagattttt aatcctagtggtcaaatttt aggcccaaaa tatgatggaa 900 aatatcttat gcaagttctt caagaaaaacttggagaaac tcgtgtgcat caagctttga 960 cagaagttgt catctcaagc tttgacatcaaaacaaataa gccagtaata ttcactaagt 1020 caaatttagc aaactctcca gaattggatgctaagatgta tgacataagt tattccacag 1080 cagcagctcc aacatatttt cctccgcattactttgttac ttaatgag 1128 251 366 PRT Artificial Synthetic polypeptide251 Asn Thr Ser Asn Gly Asp Glu Tyr Glu Phe Asn Leu Val Asp Gly Ala 1 510 15 Val Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Ile Ser Val Ala Thr 2025 30 Arg Leu Ala Gln Lys Asp Pro Ala Phe Ala Ser Ile Arg Ser Leu Asn 3540 45 Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly Thr Thr Ser Glu 5055 60 Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala Ala Thr Trp Thr Ala Val 6570 75 80 His Trp Met Leu Val Ile Gln Lys Met Thr Asp Ala Ala Ser Ser Tyr85 90 95 Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe Gln Ala Leu Asp Ser Lys100 105 110 Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Thr Gly Thr ThrThr 115 120 125 Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu ValGln Val 130 135 140 Gly Glu Asn Leu Leu Lys Lys Pro Val Ser Glu Asp AsnPro Glu Thr 145 150 155 160 Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys LeuLeu Ser Asp Arg Lys 165 170 175 Lys Leu Arg Ala Asn Lys Ala Ser Tyr GlyPro Gly Gln Leu Gly Glu 180 185 190 Met Val Thr Val Leu Ser Ile Asp GlyGly Gly Ile Arg Gly Ile Ile 195 200 205 Pro Ala Thr Ile Leu Glu Phe LeuGlu Gly Gln Leu Gln Glu Met Asp 210 215 220 Asn Asn Ala Asp Ala Arg LeuAla Asp Tyr Phe Asp Val Ile Gly Gly 225 230 235 240 Thr Ser Thr Gly GlyLeu Leu Thr Ala Met Ile Ser Thr Pro Asn Glu 245 250 255 Asn Asn Arg ProPhe Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe 260 265 270 Glu His GlyPro Gln Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro 275 280 285 Lys TyrAsp Gly Lys Tyr Leu Met Gln Val Leu Gln Glu Lys Leu Gly 290 295 300 GluThr Arg Val His Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe 305 310 315320 Asp Ile Lys Thr Asn Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala 325330 335 Asn Ser Pro Glu Leu Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr340 345 350 Ala Ala Ala Pro Thr Tyr Phe Pro Pro His Tyr Phe Val Thr 355360 365 252 55 DNA Artificial Synthetic construct 252 ggagctcgagaaaagagagg ctgaagctag ttattccaca gcagcagctc caaca 55 253 39 DNAArtificial Synthetic construct 253 ggtctagagg aattctcatt atatgtcatacatcttagc 39 254 1128 DNA Artificial Synthetic construct 254 tcgagaaaagagaggctgaa gctagttatt ccacagcagc agctccaaca tattttcctc 60 cgcattactttgttactaat actagtaatg gagatgaata tgagttcaat cttgttgatg 120 gtgctgttgctactgttgct gatccggcgt tattatccat tagcgttgca acgagacttg 180 cacaaaaggatccagcattt gcttcaatta ggtcattgaa ttacaaaaaa atgctgttgc 240 tctcattaggcactggcact acttcagagt ttgataaaac atatacagca aaagaggcag 300 ctacctggactgctgtacat tggatgttag ttatacagaa aatgactgat gcagcaagtt 360 cttacatgactgattattac ctttctactg cttttcaagc tcttgattca aaaaacaatt 420 acctcagggttcaagaaaat gcattaacag gcacaactac tgaaatggat gatgcttctg 480 aggctaatatggaattatta gtacaagttg gtgaaaactt attgaagaaa ccagtttccg 540 aagacaatcctgaaacctat gaggaagctc taaagaggtt tgcaaaattg ctctctgata 600 ggaagaaactccgagcaaac aaagcttctt atggaccagg acagttggga gaaatggtga 660 ctgttcttagtattgatgga ggtggaatta gagggatcat tccggctacc attctcgaat 720 ttcttgaaggacaacttcag gaaatggaca ataatgcaga tgcaagactt gcagattact 780 ttgatgtaattggaggaaca agtacaggag gtttattgac tgctatgata agtactccaa 840 atgaaaacaatcgacccttt gctgctgcca aagaaattgt acctttttac ttcgaacatg 900 gccctcagatttttaatcct agtggtcaaa ttttaggccc aaaatatgat ggaaaatatc 960 ttatgcaagttcttcaagaa aaacttggag aaactcgtgt gcatcaagct ttgacagaag 1020 ttgtcatctcaagctttgac atcaaaacaa ataagccagt aatattcact aagtcaaatt 1080 tagcaaactctccagaattg gatgctaaga tgtatgacat ataatgag 1128 255 366 PRT ArtificialSynthetic polypeptide 255 Ser Tyr Ser Thr Ala Ala Ala Pro Thr Tyr PhePro Pro His Tyr Phe 1 5 10 15 Val Thr Asn Thr Ser Asn Gly Asp Glu TyrGlu Phe Asn Leu Val Asp 20 25 30 Gly Ala Val Ala Thr Val Ala Asp Pro AlaLeu Leu Ser Ile Ser Val 35 40 45 Ala Thr Arg Leu Ala Gln Lys Asp Pro AlaPhe Ala Ser Ile Arg Ser 50 55 60 Leu Asn Tyr Lys Lys Met Leu Leu Leu SerLeu Gly Thr Gly Thr Thr 65 70 75 80 Ser Glu Phe Asp Lys Thr Tyr Thr AlaLys Glu Ala Ala Thr Trp Thr 85 90 95 Ala Val His Trp Met Leu Val Ile GlnLys Met Thr Asp Ala Ala Ser 100 105 110 Ser Tyr Met Thr Asp Tyr Tyr LeuSer Thr Ala Phe Gln Ala Leu Asp 115 120 125 Ser Lys Asn Asn Tyr Leu ArgVal Gln Glu Asn Ala Leu Thr Gly Thr 130 135 140 Thr Thr Glu Met Asp AspAla Ser Glu Ala Asn Met Glu Leu Leu Val 145 150 155 160 Gln Val Gly GluAsn Leu Leu Lys Lys Pro Val Ser Glu Asp Asn Pro 165 170 175 Glu Thr TyrGlu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser Asp 180 185 190 Arg LysLys Leu Arg Ala Asn Lys Ala Ser Tyr Gly Pro Gly Gln Leu 195 200 205 GlyGlu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly 210 215 220Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu Gly Gln Leu Gln Glu 225 230235 240 Met Asp Asn Asn Ala Asp Ala Arg Leu Ala Asp Tyr Phe Asp Val Ile245 250 255 Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile Ser ThrPro 260 265 270 Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala Lys Glu Ile ValPro Phe 275 280 285 Tyr Phe Glu His Gly Pro Gln Ile Phe Asn Pro Ser GlyGln Ile Leu 290 295 300 Gly Pro Lys Tyr Asp Gly Lys Tyr Leu Met Gln ValLeu Gln Glu Lys 305 310 315 320 Leu Gly Glu Thr Arg Val His Gln Ala LeuThr Glu Val Val Ile Ser 325 330 335 Ser Phe Asp Ile Lys Thr Asn Lys ProVal Ile Phe Thr Lys Ser Asn 340 345 350 Leu Ala Asn Ser Pro Glu Leu AspAla Lys Met Tyr Asp Ile 355 360 365 256 55 DNA Artificial Syntheticconstruct 256 ggagctcgag aaaagagagg ctgaagctac atatacagca aaagaggcagctacc 55 257 39 DNA Artificial Synthetic construct 257 ggtctagaggaattctcatt atttatcaaa ctctgaagt 39 258 1128 DNA Artificial Syntheticconstruct 258 tcgagaaaag agaggctgaa gctacatata cagcaaaaga ggcagctacctggactgctg 60 tacattggat gttagttata cagaaaatga ctgatgcagc aagttcttacatgactgatt 120 attacctttc tactgctttt caagctcttg attcaaaaaa caattacctcagggttcaag 180 aaaatgcatt aacaggcaca actactgaaa tggatgatgc ttctgaggctaatatggaat 240 tattagtaca agttggtgaa aacttattga agaaaccagt ttccgaagacaatcctgaaa 300 cctatgagga agctctaaag aggtttgcaa aattgctctc tgataggaagaaactccgat 360 caaacaaagc ttcttatgga ccaggacagt tgggagaaat ggtgactgttcttagtattg 420 atggaggtgg aattagaggg atcattccgg ctaccattct cgaatttcttgaaggacaac 480 ttcaggaaat ggacaataat gcagatgcaa gacttgcaga ttactttgatgtaattggag 540 gaacaagtac aggaggttta ttgactgcta tgataagtac tccaaatgaaaacaatcgac 600 cctttgctgc tgccaaagaa attgtacctt tttacttcga acatggccctcagattttta 660 atcctagtgg tcaaatttta ggcccaaaat atgatggaaa atatcttatgcaagttcttc 720 aagaaaaact tggagaaact cgtgtgcatc aagctttgac agaagttgtcatctcaagct 780 ttgacatcaa aacaaataag ccagtaatat tcactaagtc aaatttagcaaactctccag 840 aattggatgc taagatgtat gacataagtt attccacagc agcagctccaacatattttc 900 ctccgcatta ctttgttact aatactagta atggagatga atatgagttcaatcttgttg 960 atggtgctgt tgctactgtt gctgatccgg cgttattatc cattagcgttgcaacgagac 1020 ttgcacaaaa ggatccagca tttgcttcaa ttaggtcatt gaattacaaaaaaatgctgt 1080 tgctctcatt aggcactggc actacttcag agtttgataa ataatgag1128 259 366 PRT Artificial Synthetic polypeptide 259 Thr Tyr Thr AlaLys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met 1 5 10 15 Leu Val IleGln Lys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp 20 25 30 Tyr Tyr LeuSer Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn Tyr 35 40 45 Leu Arg ValGln Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp 50 55 60 Asp Ala SerGlu Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn 65 70 75 80 Leu LeuLys Lys Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu 85 90 95 Ala LeuLys Arg Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg 100 105 110 SerAsn Lys Ala Ser Tyr Gly Pro Gly Gln Leu Gly Glu Met Val Thr 115 120 125Val Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr 130 135140 Ile Leu Glu Phe Leu Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala 145150 155 160 Asp Ala Arg Leu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr SerThr 165 170 175 Gly Gly Leu Leu Thr Ala Met Ile Ser Thr Pro Asn Glu AsnAsn Arg 180 185 190 Pro Phe Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr PheGlu His Gly 195 200 205 Pro Gln Ile Phe Asn Pro Ser Gly Gln Ile Leu GlyPro Lys Tyr Asp 210 215 220 Gly Lys Tyr Leu Met Gln Val Leu Gln Glu LysLeu Gly Glu Thr Arg 225 230 235 240 Val His Gln Ala Leu Thr Glu Val ValIle Ser Ser Phe Asp Ile Lys 245 250 255 Thr Asn Lys Pro Val Ile Phe ThrLys Ser Asn Leu Ala Asn Ser Pro 260 265 270 Glu Leu Asp Ala Lys Met TyrAsp Ile Ser Tyr Ser Thr Ala Ala Ala 275 280 285 Pro Thr Tyr Phe Pro ProHis Tyr Phe Val Thr Asn Thr Ser Asn Gly 290 295 300 Asp Glu Tyr Glu PheAsn Leu Val Asp Gly Ala Val Ala Thr Val Ala 305 310 315 320 Asp Pro AlaLeu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys 325 330 335 Asp ProAla Phe Ala Ser Ile Arg Ser Leu Asn Tyr Lys Lys Met Leu 340 345 350 LeuLeu Ser Leu Gly Thr Gly Thr Thr Ser Glu Phe Asp Lys 355 360 365 260 55DNA Artificial Synthetic construct 260 ggagctcgag aaaagagagg ctgaagctaatgcattaaca ggcacaacta ctgaa 55 261 39 DNA Artificial Synthetic construct261 ggtctagagg aattctcatt attcttgaac cctgaggta 39 262 1128 DNAArtificial Synthetic construct 262 tcgagaaaag agaggctgaa gctaatgcattaacaggcac aactactgaa atggatgatg 60 cttctgaggc taatatggaa ttattagtacaagttggtga aaacttattg aagaaaccag 120 tttccgaaga caatcctgaa acctatgaggaagctctaaa gaggtttgca aaattgctct 180 ctgataggaa gaaactccga gcaaacaaagcttcttatgg accaggacag ttgggagaaa 240 tggtgactgt tcttagtatt gatggaggtggaattagagg gatcattccg gctaccattc 300 tcgaatttct tgaaggacaa cttcaggaaatggacaataa tgcagatgca agacttgcag 360 attactttga tgtaattgga ggaacaagtacaggaggttt attgactgct atgataagta 420 ctccaaatga aaacaatcga ccctttgctgctgccaaaga aattgtacct ttttacttcg 480 aacatggccc tcagattttt aatcctagtggtcaaatttt aggcccaaaa tatgatggaa 540 aatatcttat gcaagttctt caagaaaaacttggagaaac tcgtgtgcat caagctttga 600 cagaagttgt catctcaagc tttgacatcaaaacaaataa gccagtaata ttcactaagt 660 caaatttagc aaactctcca gaattggatgctaagatgta tgacataagt tattccacag 720 cagcagctcc aacatatttt cctccgcattactttgttac taatactagt aatggagatg 780 aatatgagtt caatcttgtt gatggtgctgttgctactgt tgctgatccg gcgttattat 840 ccattagcgt tgcaacgaga cttgcacaaaaggatccagc atttgcttca attaggtcat 900 tgaattacaa aaaaatgctg ttgctctcattaggcactgg cactacttca gagtttgata 960 aaacatatac agcaaaagag gcagctacctggactgctgt acattggatg ttagttatac 1020 agaaaatgac tgatgcagca agttcttacatgactgatta ttacctttct actgcttttc 1080 aagctcttga ttcaaaaaac aattacctcagggttcaaga ataatgag 1128 263 366 PRT Artificial Synthetic polypeptide263 Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu Ala 1 510 15 Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys Pro 2025 30 Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe 3540 45 Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser 5055 60 Tyr Gly Pro Gly Gln Leu Gly Glu Met Val Thr Val Leu Ser Ile Asp 6570 75 80 Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu Glu Phe Leu85 90 95 Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala Asp Ala Arg Leu Ala100 105 110 Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly Leu LeuThr 115 120 125 Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe AlaAla Ala 130 135 140 Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro GlnIle Phe Asn 145 150 155 160 Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr AspGly Lys Tyr Leu Met 165 170 175 Gln Val Leu Gln Glu Lys Leu Gly Glu ThrArg Val His Gln Ala Leu 180 185 190 Thr Glu Val Val Ile Ser Ser Phe AspIle Lys Thr Asn Lys Pro Val 195 200 205 Ile Phe Thr Lys Ser Asn Leu AlaAsn Ser Pro Glu Leu Asp Ala Lys 210 215 220 Met Tyr Asp Ile Ser Tyr SerThr Ala Ala Ala Pro Thr Tyr Phe Pro 225 230 235 240 Pro His Tyr Phe ValThr Asn Thr Ser Asn Gly Asp Glu Tyr Glu Phe 245 250 255 Asn Leu Val AspGly Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu 260 265 270 Ser Ile SerVal Ala Thr Arg Leu Ala Gln Lys Asp Pro Ala Phe Ala 275 280 285 Ser IleArg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly 290 295 300 ThrGly Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr Ala Lys Glu Ala 305 310 315320 Ala Thr Trp Thr Ala Val His Trp Met Leu Val Ile Gln Lys Met Thr 325330 335 Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr Ala Phe340 345 350 Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln Glu 355360 365 264 1158 DNA Artificial Synthetic construct 264 atggccaccaccaagagctt cctcatcctg atcttcatga tcctggccac caccagcagc 60 accttcgcccagctcggcga gatggtgacc gtgctctcca tcgacggcgg tggcatcagg 120 ggcatcatcccggccaccat cctggagttc ctggagggcc aactccagga gatggacaac 180 aacgccgacgcccgcctggc cgactacttc gacgtgatcg gtggcaccag caccggcggt 240 ctcctgaccgccatgatctc cactccgaac gagaacaacc gccccttcgc cgctgcgaag 300 gagatcgtcccgttctactt cgaacacggc cctcagattt tcaacccctc gggtcaaatc 360 ctgggccccaagtacgacgg caagtacctt atgcaagtgc ttcaggagaa gctgggcgag 420 actagggtgcaccaggcgct gaccgaggtc gtcatctcca gcttcgacat caagaccaac 480 aagccagtcatcttcaccaa gtccaacctg gccaacagcc cggagctgga cgctaagatg 540 tacgacatctcctactccac tgctgccgct cccacgtact tccctccgca ctacttcgtc 600 accaacaccagcaacggcga cgagtacgag ttcaaccttg ttgacggtgc ggtggctacg 660 gtggcggacccggcgctcct gtccatcagc gtcgccacgc gcctggccca gaaggatcca 720 gccttcgctagcattaggag cctcaactac aagaagatgc tgctgctcag cctgggcact 780 ggcacgacctccgagttcga caagacctac actgccaagg aggccgctac ctggaccgcc 840 gtccattggatgctggtcat ccagaagatg acggacgccg cttccagcta catgaccgac 900 tactacctctccactgcgtt ccaggcgctt gactccaaga acaactacct ccgtgttcag 960 gagaatgccctcactggcac cacgaccgag atggacgatg cctccgaggc caacatggag 1020 ctgctcgtccaggtgggtga gaacctcctg aagaagcccg tctccgaaga caatcccgag 1080 acctatgaggaagcgctcaa gcgctttgcc aagctgctct ctgataggaa gaaactccgc 1140 gctaacaaggccagctac 1158 265 386 PRT Artificial Synthetic polypeptide 265 Met AlaThr Thr Lys Ser Phe Leu Ile Leu Ile Phe Met Ile Leu Ala 1 5 10 15 ThrThr Ser Ser Thr Phe Ala Gln Leu Gly Glu Met Val Thr Val Leu 20 25 30 SerIle Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu 35 40 45 GluPhe Leu Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala Asp Ala 50 55 60 ArgLeu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly 65 70 75 80Leu Leu Thr Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe 85 90 95Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro Gln 100 105110 Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys 115120 125 Tyr Leu Met Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His130 135 140 Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys ThrAsn 145 150 155 160 Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Asn SerPro Glu Leu 165 170 175 Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr AlaAla Ala Pro Thr 180 185 190 Tyr Phe Pro Pro His Tyr Phe Val Thr Asn ThrSer Asn Gly Asp Glu 195 200 205 Tyr Glu Phe Asn Leu Val Asp Gly Ala ValAla Thr Val Ala Asp Pro 210 215 220 Ala Leu Leu Ser Ile Ser Val Ala ThrArg Leu Ala Gln Lys Asp Pro 225 230 235 240 Ala Phe Ala Ser Ile Arg SerLeu Asn Tyr Lys Lys Met Leu Leu Leu 245 250 255 Ser Leu Gly Thr Gly ThrThr Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260 265 270 Lys Glu Ala Ala ThrTrp Thr Ala Val His Trp Met Leu Val Ile Gln 275 280 285 Lys Met Thr AspAla Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser 290 295 300 Thr Ala PheGln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln 305 310 315 320 GluAsn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu 325 330 335Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys 340 345350 Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg 355360 365 Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala370 375 380 Ser Tyr 385 266 55 DNA Artificial Synthetic construct 266ggagctcgag aaaagagagg ctgaagctag cctcaactac aagaagatgc tgctg 55 267 42DNA Artificial Synthetic construct 267 gccgagctgt cctggtccgt agctggccttgttagcgcgg ag 42 268 36 DNA Artificial Synthetic construct 268gccagctacg gaccaggaca gctcggcgag atggtg 36 269 39 DNA ArtificialSynthetic construct 269 ggtctagagg aattctcatt acctaatgct agcgaaggc 39270 1167 DNA Artificial Synthetic construct 270 atggccacca ccaagagcttcctcatcctg atcttcatga tcctggccac caccagcagc 60 accttcgcca gcctcaactacaagaagatg ctgctgctca gcctgggcac tggcacgacc 120 tccgagttcg acaagacctacactgccaag gaggccgcta cctggaccgc cgtccattgg 180 atgctggtca tccagaagatgacggacgcc gcttccagct acatgaccga ctactacctc 240 tccactgcgt tccaggcgcttgactccaag aacaactacc tccgtgttca ggagaatgcc 300 ctcactggca ccacgaccgagatggacgat gcctccgagg ccaacatgga gctgctcgtc 360 caggtgggtg agaacctcctgaagaagccc gtctccgaag acaatcccga gacctatgag 420 gaagcgctca agcgctttgccaagctgctc tctgatagga agaaactccg cgctaacaag 480 gccagctacg gaccaggacagctcggcgag atggtgaccg tgctctccat cgacggcggt 540 ggcatcaggg gcatcatcccggccaccatc ctggagttcc tggagggcca actccaggag 600 atggacaaca acgccgacgcccgcctggcc gactacttcg acgtgatcgg tggcaccagc 660 accggcggtc tcctgaccgccatgatctcc actccgaacg agaacaaccg ccccttcgcc 720 gctgcgaagg agatcgtcccgttctacttc gaacacggcc ctcagatttt caacccctcg 780 ggtcaaatcc tgggccccaagtacgacggc aagtacctta tgcaagtgct tcaggagaag 840 ctgggcgaga ctagggtgcaccaggcgctg accgaggtcg tcatctccag cttcgacatc 900 aagaccaaca agccagtcatcttcaccaag tccaacctgg ccaacagccc ggagctggac 960 gctaagatgt acgacatctcctactccact gctgccgctc ccacgtactt ccctccgcac 1020 tacttcgtca ccaacaccagcaacggcgac gagtacgagt tcaaccttgt tgacggtgcg 1080 gtggctacgg tggcggacccggcgctcctg tccatcagcg tcgccacgcg cctggcccag 1140 aaggatccag ccttcgctagcattagg 1167 271 389 PRT Artificial Synthetic polypeptide 271 Met AlaThr Thr Lys Ser Phe Leu Ile Leu Ile Phe Met Ile Leu Ala 1 5 10 15 ThrThr Ser Ser Thr Phe Ala Ser Leu Asn Tyr Lys Lys Met Leu Leu 20 25 30 LeuSer Leu Gly Thr Gly Thr Thr Ser Glu Phe Asp Lys Thr Tyr Thr 35 40 45 AlaLys Glu Ala Ala Thr Trp Thr Ala Val His Trp Met Leu Val Ile 50 55 60 GlnLys Met Thr Asp Ala Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu 65 70 75 80Ser Thr Ala Phe Gln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val 85 90 95Gln Glu Asn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser 100 105110 Glu Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys 115120 125 Lys Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys130 135 140 Arg Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala AsnLys 145 150 155 160 Ala Ser Tyr Gly Pro Gly Gln Leu Gly Glu Met Val ThrVal Leu Ser 165 170 175 Ile Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro AlaThr Ile Leu Glu 180 185 190 Phe Leu Glu Gly Gln Leu Gln Glu Met Asp AsnAsn Ala Asp Ala Arg 195 200 205 Leu Ala Asp Tyr Phe Asp Val Ile Gly GlyThr Ser Thr Gly Gly Leu 210 215 220 Leu Thr Ala Met Ile Ser Thr Pro AsnGlu Asn Asn Arg Pro Phe Ala 225 230 235 240 Ala Ala Lys Glu Ile Val ProPhe Tyr Phe Glu His Gly Pro Gln Ile 245 250 255 Phe Asn Pro Ser Gly GlnIle Leu Gly Pro Lys Tyr Asp Gly Lys Tyr 260 265 270 Leu Met Gln Val LeuGln Glu Lys Leu Gly Glu Thr Arg Val His Gln 275 280 285 Ala Leu Thr GluVal Val Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys 290 295 300 Pro Val IlePhe Thr Lys Ser Asn Leu Ala Asn Ser Pro Glu Leu Asp 305 310 315 320 AlaLys Met Tyr Asp Ile Ser Tyr Ser Thr Ala Ala Ala Pro Thr Tyr 325 330 335Phe Pro Pro His Tyr Phe Val Thr Asn Thr Ser Asn Gly Asp Glu Tyr 340 345350 Glu Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Ala Asp Pro Ala 355360 365 Leu Leu Ser Ile Ser Val Ala Thr Arg Leu Ala Gln Lys Asp Pro Ala370 375 380 Phe Ala Ser Ile Arg 385 272 55 DNA Artificial Syntheticconstruct 272 ggagctcgag aaaagagagg ctgaagctac tgccaaggag gccgctacctggacc 55 273 39 DNA Artificial Synthetic construct 273 ggtctagaggaattctcatt acttgtcgaa ctcggaggt 39 274 1167 DNA Artificial Syntheticconstruct 274 atggccacca ccaagagctt cctcatcctg atcttcatga tcctggccaccaccagcagc 60 accttcgcca cctacactgc caaggaggcc gctacctgga ccgccgtccattggatgctg 120 gtcatccaga agatgacgga cgccgcttcc agctacatga ccgactactacctctccact 180 gcgttccagg cgcttgactc caagaacaac tacctccgtg ttcaggagaatgccctcact 240 ggcaccacga ccgagatgga cgatgcctcc gaggccaaca tggagctgctcgtccaggtg 300 ggtgagaacc tcctgaagaa gcccgtctcc gaagacaatc ccgagacctatgaggaagcg 360 ctcaagcgct ttgccaagct gctctctgat aggaagaaac tccgcgctaacaaggccagc 420 tacggaccag gacagctcgg cgagatggtg accgtgctct ccatcgacggcggtggcatc 480 aggggcatca tcccggccac catcctggag ttcctggagg gccaactccaggagatggac 540 aacaacgccg acgcccgcct ggccgactac ttcgacgtga tcggtggcaccagcaccggc 600 ggtctcctga ccgccatgat ctccactccg aacgagaaca accgccccttcgccgctgcg 660 aaggagatcg tcccgttcta cttcgaacac ggccctcaga ttttcaacccctcgggtcaa 720 atcctgggcc ccaagtacga cggcaagtac cttatgcaag tgcttcaggagaagctgggc 780 gagactaggg tgcaccaggc gctgaccgag gtcgtcatct ccagcttcgacatcaagacc 840 aacaagccag tcatcttcac caagtccaac ctggccaaca gcccggagctggacgctaag 900 atgtacgaca tctcctactc cactgctgcc gctcccacgt acttccctccgcactacttc 960 gtcaccaaca ccagcaacgg cgacgagtac gagttcaacc ttgttgacggtgcggtggct 1020 acggtggcgg acccggcgct cctgtccatc agcgtcgcca cgcgcctggcccagaaggat 1080 ccagccttcg ctagcattag gagcctcaac tacaagaaga tgctgctgctcagcctgggc 1140 actggcacga cctccgagtt cgacaag 1167 275 389 PRTArtificial Synthetic polypeptide 275 Met Ala Thr Thr Lys Ser Phe Leu IleLeu Ile Phe Met Ile Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr Phe Ala ThrTyr Thr Ala Lys Glu Ala Ala Thr 20 25 30 Trp Thr Ala Val His Trp Met LeuVal Ile Gln Lys Met Thr Asp Ala 35 40 45 Ala Ser Ser Tyr Met Thr Asp TyrTyr Leu Ser Thr Ala Phe Gln Ala 50 55 60 Leu Asp Ser Lys Asn Asn Tyr LeuArg Val Gln Glu Asn Ala Leu Thr 65 70 75 80 Gly Thr Thr Thr Glu Met AspAsp Ala Ser Glu Ala Asn Met Glu Leu 85 90 95 Leu Val Gln Val Gly Glu AsnLeu Leu Lys Lys Pro Val Ser Glu Asp 100 105 110 Asn Pro Glu Thr Tyr GluGlu Ala Leu Lys Arg Phe Ala Lys Leu Leu 115 120 125 Ser Asp Arg Lys LysLeu Arg Ala Asn Lys Ala Ser Tyr Gly Pro Gly 130 135 140 Gln Leu Gly GluMet Val Thr Val Leu Ser Ile Asp Gly Gly Gly Ile 145 150 155 160 Arg GlyIle Ile Pro Ala Thr Ile Leu Glu Phe Leu Glu Gly Gln Leu 165 170 175 GlnGlu Met Asp Asn Asn Ala Asp Ala Arg Leu Ala Asp Tyr Phe Asp 180 185 190Val Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile Ser 195 200205 Thr Pro Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala Lys Glu Ile Val 210215 220 Pro Phe Tyr Phe Glu His Gly Pro Gln Ile Phe Asn Pro Ser Gly Gln225 230 235 240 Ile Leu Gly Pro Lys Tyr Asp Gly Lys Tyr Leu Met Gln ValLeu Gln 245 250 255 Glu Lys Leu Gly Glu Thr Arg Val His Gln Ala Leu ThrGlu Val Val 260 265 270 Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys Pro ValIle Phe Thr Lys 275 280 285 Ser Asn Leu Ala Asn Ser Pro Glu Leu Asp AlaLys Met Tyr Asp Ile 290 295 300 Ser Tyr Ser Thr Ala Ala Ala Pro Thr TyrPhe Pro Pro His Tyr Phe 305 310 315 320 Val Thr Asn Thr Ser Asn Gly AspGlu Tyr Glu Phe Asn Leu Val Asp 325 330 335 Gly Ala Val Ala Thr Val AlaAsp Pro Ala Leu Leu Ser Ile Ser Val 340 345 350 Ala Thr Arg Leu Ala GlnLys Asp Pro Ala Phe Ala Ser Ile Arg Ser 355 360 365 Leu Asn Tyr Lys LysMet Leu Leu Leu Ser Leu Gly Thr Gly Thr Thr 370 375 380 Ser Glu Phe AspLys 385 276 7 PRT Artificial Synthetic polypeptide 276 Gly Gly Gly SerGly Gly Gly 1 5 277 3 PRT Artificial Synthetic polypeptide 277 Gly ProGly 1 278 386 PRT Solanum tuberosum 278 Met Ala Thr Thr Lys Ser Phe LeuIle Leu Phe Phe Met Ile Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr Cys AlaLys Leu Glu Glu Met Val Thr Val Leu 20 25 30 Ser Ile Asp Gly Gly Gly IleLys Gly Ile Ile Pro Ala Ile Ile Leu 35 40 45 Glu Phe Leu Glu Gly Gln LeuGln Glu Val Asp Asn Asn Lys Asp Ala 50 55 60 Arg Leu Ala Asp Tyr Phe AspVal Ile Gly Gly Thr Ser Thr Gly Gly 65 70 75 80 Leu Leu Thr Ala Met IleThr Thr Pro Asn Glu Asn Asn Arg Pro Phe 85 90 95 Ala Ala Ala Lys Asp IleVal Pro Phe Tyr Phe Glu His Gly Pro His 100 105 110 Ile Phe Asn Tyr SerGly Ser Ile Ile Gly Pro Met Tyr Asp Gly Lys 115 120 125 Tyr Leu Leu GlnVal Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His 130 135 140 Gln Ala LeuThr Glu Val Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn 145 150 155 160 LysPro Val Ile Phe Thr Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu 165 170 175Asp Ala Lys Met Tyr Asp Ile Cys Tyr Ser Thr Ala Ala Ala Pro Ile 180 185190 Tyr Phe Pro Pro His Tyr Phe Ile Thr His Thr Ser Asn Gly Asp Ile 195200 205 Tyr Glu Phe Asn Leu Val Asp Gly Gly Val Ala Thr Val Gly Asp Pro210 215 220 Ala Leu Leu Ser Leu Ser Val Ala Thr Arg Leu Ala Gln Glu AspPro 225 230 235 240 Ala Phe Ser Ser Ile Lys Ser Leu Asp Tyr Lys Gln MetLeu Leu Leu 245 250 255 Ser Leu Gly Thr Gly Thr Asn Ser Glu Phe Asp LysThr Tyr Thr Ala 260 265 270 Gln Glu Ala Ala Lys Trp Gly Pro Leu Arg TrpMet Leu Ala Ile Gln 275 280 285 Gln Met Thr Asn Ala Ala Ser Ser Tyr MetThr Asp Tyr Tyr Ile Ser 290 295 300 Thr Val Phe Gln Ala Arg His Ser GlnAsn Asn Tyr Leu Arg Val Gln 305 310 315 320 Glu Asn Ala Leu Thr Gly ThrThr Thr Glu Met Asp Asp Ala Ser Glu 325 330 335 Ala Asn Met Glu Leu LeuVal Gln Val Gly Glu Thr Leu Leu Lys Lys 340 345 350 Pro Val Ser Lys AspSer Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg 355 360 365 Phe Ala Lys LeuLeu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370 375 380 Ser Tyr 385279 386 PRT Solanum tuberosum 279 Met Ala Thr Thr Lys Ser Val Leu ValLeu Phe Phe Met Ile Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr Cys Ala ThrLeu Gly Glu Met Val Thr Val Leu 20 25 30 Ser Ile Asp Gly Gly Gly Ile LysGly Ile Ile Pro Ala Thr Ile Leu 35 40 45 Glu Phe Leu Glu Gly Gln Leu GlnGlu Val Asp Asn Asn Lys Asp Ala 50 55 60 Arg Leu Ala Asp Tyr Phe Asp ValIle Gly Gly Thr Ser Thr Gly Gly 65 70 75 80 Leu Leu Thr Ala Met Ile ThrThr Pro Asn Glu Asn Asn Arg Pro Phe 85 90 95 Ala Ala Ala Lys Asp Ile ValPro Phe Tyr Phe Glu His Gly Pro His 100 105 110 Ile Phe Asn Ser Ser GlySer Ile Phe Gly Pro Met Tyr Asp Gly Lys 115 120 125 Tyr Phe Leu Gln ValLeu Gln Glu Lys Leu Gly Glu Thr Arg Val His 130 135 140 Gln Ala Leu ThrGlu Val Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn 145 150 155 160 Lys ProVal Ile Phe Thr Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu 165 170 175 AspAla Lys Met Asn Asp Ile Cys Tyr Ser Thr Ala Ala Ala Pro Thr 180 185 190Tyr Phe Pro Pro His Tyr Phe Val Thr His Thr Ser Asn Gly Asp Lys 195 200205 Tyr Glu Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Gly Asp Pro 210215 220 Ala Leu Leu Ser Leu Ser Val Arg Thr Lys Leu Ala Gln Val Asp Pro225 230 235 240 Lys Phe Ala Ser Ile Lys Ser Leu Asn Tyr Asn Glu Met LeuLeu Leu 245 250 255 Ser Leu Gly Thr Gly Thr Asn Ser Glu Phe Asp Lys ThrTyr Thr Ala 260 265 270 Glu Glu Ala Ala Lys Trp Gly Pro Leu Arg Trp IleLeu Ala Ile Gln 275 280 285 Gln Met Thr Asn Ala Ala Ser Ser Tyr Met ThrAsp Tyr Tyr Leu Ser 290 295 300 Thr Val Phe Gln Ala Arg His Ser Gln AsnAsn Tyr Leu Arg Val Gln 305 310 315 320 Glu Asn Ala Leu Thr Gly Thr ThrThr Glu Met Asp Asp Ala Ser Glu 325 330 335 Ala Asn Met Glu Leu Leu ValGln Val Gly Glu Lys Leu Leu Lys Lys 340 345 350 Pro Val Ser Lys Asp SerPro Glu Thr Tyr Glu Glu Ala Leu Lys Arg 355 360 365 Phe Ala Lys Leu LeuSer Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370 375 380 Ser Tyr 385 280365 PRT Solanum tuberosum 280 Met Ala Leu Glu Glu Met Val Ala Val LeuSer Ile Asp Gly Gly Gly 1 5 10 15 Ile Lys Gly Ile Ile Pro Gly Thr IleLeu Glu Phe Leu Glu Gly Gln 20 25 30 Leu Gln Lys Met Asp Asn Asn Ala AspAla Arg Leu Ala Asp Tyr Phe 35 40 45 Asp Val Ile Gly Gly Thr Ser Thr GlyGly Leu Leu Thr Ala Met Ile 50 55 60 Thr Thr Pro Asn Glu Asn Asn Arg ProPhe Ala Ala Ala Asn Glu Ile 65 70 75 80 Val Pro Phe Tyr Phe Glu His GlyPro His Ile Phe Asn Ser Arg Tyr 85 90 95 Trp Pro Ile Phe Trp Pro Lys TyrAsp Gly Lys Tyr Leu Met Gln Val 100 105 110 Leu Gln Glu Lys Leu Gly GluThr Arg Val His Gln Ala Leu Thr Glu 115 120 125 Val Ala Ile Ser Ser PheAsp Ile Lys Thr Asn Lys Pro Val Ile Phe 130 135 140 Thr Lys Ser Asn LeuAla Lys Ser Pro Glu Leu Asp Ala Lys Thr Tyr 145 150 155 160 Asp Ile CysTyr Ser Thr Ala Ala Ala Pro Thr Tyr Phe Pro Pro His 165 170 175 Tyr PheAla Thr Asn Thr Ile Asn Gly Asp Lys Tyr Glu Phe Asn Leu 180 185 190 ValAsp Gly Ala Val Ala Thr Val Ala Asp Pro Ala Leu Leu Ser Val 195 200 205Ser Val Ala Thr Arg Arg Ala Gln Glu Asp Pro Ala Phe Ala Ser Ile 210 215220 Arg Ser Leu Asn Tyr Lys Lys Met Leu Leu Leu Ser Leu Gly Thr Gly 225230 235 240 Thr Thr Ser Glu Phe Asp Lys Thr His Thr Ala Glu Glu Thr AlaLys 245 250 255 Trp Gly Ala Leu Gln Trp Met Leu Val Ile Gln Gln Met ThrGlu Ala 260 265 270 Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser Thr ValPhe Gln Asp 275 280 285 Leu His Ser Gln Asn Asn Tyr Leu Arg Val Gln GluAsn Ala Leu Thr 290 295 300 Gly Thr Thr Thr Lys Ala Asp Asp Ala Ser GluAla Asn Met Glu Leu 305 310 315 320 Leu Ala Gln Val Gly Glu Asn Leu LeuLys Lys Pro Val Ser Lys Asp 325 330 335 Asn Pro Glu Thr Tyr Glu Glu AlaLeu Lys Arg Phe Ala Lys Leu Leu 340 345 350 Ser Asp Arg Lys Lys Leu ArgAla Asn Lys Ala Ser Tyr 355 360 365 281 364 PRT Solanum tuberosum 281Pro Trp Leu Glu Glu Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly 1 5 1015 Ile Lys Gly Ile Ile Pro Ala Ile Ile Leu Glu Phe Leu Glu Gly Gln 20 2530 Leu Gln Glu Val Asp Asn Asn Lys Asp Ala Arg Leu Ala Asp Tyr Phe 35 4045 Asp Val Ile Gly Gly Thr Ser Thr Gly Gly Leu Leu Thr Ala Met Ile 50 5560 Thr Thr Pro Asn Glu Asn Asn Arg Pro Phe Ala Ala Ala Lys Asp Ile 65 7075 80 Val Pro Phe Tyr Phe Glu His Gly Pro His Ile Phe Asn Tyr Ser Gly 8590 95 Ser Ile Leu Gly Pro Met Tyr Asp Gly Lys Tyr Leu Leu Gln Val Leu100 105 110 Gln Glu Lys Leu Gly Glu Thr Arg Val His Gln Ala Leu Thr GluVal 115 120 125 Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn Lys Pro Val IlePhe Thr 130 135 140 Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu Asp Ala LysMet Tyr Asp 145 150 155 160 Ile Cys Tyr Ser Thr Ala Ala Ala Pro Ile TyrPhe Pro Pro His His 165 170 175 Phe Val Thr His Thr Ser Asn Gly Ala ArgTyr Glu Phe Asn Leu Val 180 185 190 Asp Gly Ala Val Ala Thr Val Gly AspPro Ala Leu Leu Ser Leu Ser 195 200 205 Val Ala Thr Arg Leu Ala Gln GluAsp Pro Ala Phe Ser Ser Ile Lys 210 215 220 Ser Leu Asp Tyr Lys Gln MetLeu Leu Leu Ser Leu Gly Thr Gly Thr 225 230 235 240 Asn Ser Glu Phe AspLys Thr Tyr Thr Ala Glu Glu Ala Ala Lys Trp 245 250 255 Gly Pro Leu ArgTrp Met Leu Ala Ile Gln Gln Met Thr Asn Ala Ala 260 265 270 Ser Phe TyrMet Thr Asp Tyr Tyr Ile Ser Thr Val Phe Gln Ala Arg 275 280 285 His SerGln Asn Asn Tyr Leu Arg Val Gln Glu Asn Ala Leu Asn Gly 290 295 300 ThrThr Thr Glu Met Asp Asp Ala Ser Glu Ala Asn Met Glu Leu Leu 305 310 315320 Val Gln Val Gly Glu Thr Leu Leu Lys Lys Pro Val Ser Arg Asp Ser 325330 335 Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg Phe Ala Lys Leu Leu Ser340 345 350 Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala Ser Tyr 355 360 282386 PRT Solanum tuberosum 282 Met Ala Thr Thr Lys Ser Phe Leu Ile LeuPhe Phe Met Ile Leu Ala 1 5 10 15 Thr Thr Ser Ser Thr Cys Ala Lys LeuGlu Glu Met Val Thr Val Leu 20 25 30 Ser Ile Asp Gly Gly Gly Ile Lys GlyIle Ile Pro Ala Ile Ile Leu 35 40 45 Glu Phe Leu Glu Gly Gln Leu Gln GluVal Asp Asn Asn Lys Asp Ala 50 55 60 Arg Leu Ala Asp Tyr Phe Asp Val IleGly Gly Thr Ser Thr Gly Gly 65 70 75 80 Leu Leu Thr Ala Met Ile Thr ThrPro Asn Glu Asn Asn Arg Pro Phe 85 90 95 Ala Ala Ala Lys Asp Ile Val ProPhe Tyr Phe Glu His Gly Pro His 100 105 110 Ile Phe Asn Tyr Ser Gly SerIle Leu Gly Pro Met Tyr Asp Gly Lys 115 120 125 Tyr Leu Leu Gln Val LeuGln Glu Lys Leu Gly Glu Thr Arg Val His 130 135 140 Gln Ala Leu Thr GluVal Ala Ile Ser Ser Phe Asp Ile Lys Thr Asn 145 150 155 160 Lys Pro ValIle Phe Thr Lys Ser Asn Leu Ala Lys Ser Pro Glu Leu 165 170 175 Asp AlaLys Met Tyr Asp Ile Cys Tyr Ser Thr Ala Ala Ala Pro Ile 180 185 190 TyrPhe Pro Pro His His Phe Val Thr His Thr Ser Asn Gly Ala Arg 195 200 205Tyr Glu Phe Asn Leu Val Asp Gly Ala Val Ala Thr Val Gly Asp Pro 210 215220 Ala Leu Leu Ser Leu Ser Val Ala Thr Arg Leu Ala Gln Glu Asp Pro 225230 235 240 Ala Phe Ser Ser Ile Lys Ser Leu Asp Tyr Lys Gln Met Leu LeuLeu 245 250 255 Ser Leu Gly Thr Gly Thr Asn Ser Glu Phe Asp Lys Thr TyrThr Ala 260 265 270 Glu Glu Ala Ala Lys Trp Gly Pro Leu Arg Trp Met LeuAla Ile Gln 275 280 285 Gln Met Thr Asn Ala Ala Ser Ser Tyr Met Thr AspTyr Tyr Ile Ser 290 295 300 Thr Val Phe Gln Ala Arg His Ser Gln Asn AsnTyr Leu Arg Val Gln 305 310 315 320 Glu Asn Ala Leu Asn Gly Thr Thr ThrGlu Met Asp Asp Ala Ser Glu 325 330 335 Ala Asn Met Glu Leu Leu Val GlnVal Gly Ala Thr Leu Leu Lys Lys 340 345 350 Pro Val Ser Lys Asp Ser ProGlu Thr Tyr Glu Glu Ala Leu Lys Arg 355 360 365 Phe Ala Lys Leu Leu SerAsp Arg Lys Lys Leu Arg Ala Asn Lys Ala 370 375 380 Ser Tyr 385 283 10PRT Artificial Synthetic polypeptide 283 Ala Phe Phe Asp Lys Thr Tyr ThrAla Lys 1 5 10 284 10 PRT Artificial Synthetic polypeptide 284 Cys IlePhe Asp Ser Thr Tyr Thr Ala Lys 1 5 10 285 1161 DNA Solanum tuberosum285 atggcaacta ctaaatcttt tttaatttta atatttatga tattagcaac tactagttca 60acatttgctc agttgggaga aatggtgact gttcttagta ttgatggagg tggaattaga 120gggatcattc cggctaccat tctcgaattt cttgaaggac aacttcagga aatggacaat 180aatgcagatg caagacttgc agattacttt gatgtaattg gaggaacaag tacaggaggt 240ttattgactg ctatgataag tactccaaat gaaaacaatc gaccctttgc tgctgccaaa 300gaaattgtac ctttttactt cgaacatggc cctcagattt ttaatcctag tggtcaaatt 360ttaggcccaa aatatgatgg aaaatatctt atgcaagttc ttcaagaaaa acttggagaa 420actcgtgtgc atcaagcttt gacagaagtt gtcatctcaa gctttgacat caaaacaaat 480aagccagtaa tattcactaa gtcaaattta gcaaactctc cagaattgga tgctaagatg 540tatgacataa gttattccac agcagcagct ccaacatatt ttcctccgca ttactttgtt 600actaatacta gtaatggaga tgaatatgag ttcaatcttg ttgatggtgc tgttgctact 660gttgctgatc cggcgttatt atccattagc gttgcaacga gacttgcaca aaaggatcca 720gcatttgctt caattaggtc attgaattac aaaaaaatgc tgttgctctc attaggcact 780ggcactactt cagagtttga taaaacatat acagcaaaag aggcagctac ctggactgct 840gtacattgga tgttagttat acagaaaatg actgatgcag caagttctta catgactgat 900tattaccttt ctactgcttt tcaagctctt gattcaaaaa acaattacct cagggttcaa 960gaaaatgcat taacaggcac aactactgaa atggatgatg cttctgaggc taatatggaa 1020ttattagtac aagttggtga aaacttattg aagaaaccag tttccgaaga caatcctgaa 1080acctatgagg aagctctaaa gaggtttgca aaattgctct ctgataggaa gaaactccga 1140gcaaacaaag cttcttatta a 1161 286 386 PRT Solanum tuberosum 286 Met AlaThr Thr Lys Ser Phe Leu Ile Leu Ile Phe Met Ile Leu Ala 1 5 10 15 ThrThr Ser Ser Thr Phe Ala Gln Leu Gly Glu Met Val Thr Val Leu 20 25 30 SerIle Asp Gly Gly Gly Ile Arg Gly Ile Ile Pro Ala Thr Ile Leu 35 40 45 GluPhe Leu Glu Gly Gln Leu Gln Glu Met Asp Asn Asn Ala Asp Ala 50 55 60 ArgLeu Ala Asp Tyr Phe Asp Val Ile Gly Gly Thr Ser Thr Gly Gly 65 70 75 80Leu Leu Thr Ala Met Ile Ser Thr Pro Asn Glu Asn Asn Arg Pro Phe 85 90 95Ala Ala Ala Lys Glu Ile Val Pro Phe Tyr Phe Glu His Gly Pro Gln 100 105110 Ile Phe Asn Pro Ser Gly Gln Ile Leu Gly Pro Lys Tyr Asp Gly Lys 115120 125 Tyr Leu Met Gln Val Leu Gln Glu Lys Leu Gly Glu Thr Arg Val His130 135 140 Gln Ala Leu Thr Glu Val Val Ile Ser Ser Phe Asp Ile Lys ThrAsn 145 150 155 160 Lys Pro Val Ile Phe Thr Lys Ser Asn Leu Ala Asn SerPro Glu Leu 165 170 175 Asp Ala Lys Met Tyr Asp Ile Ser Tyr Ser Thr AlaAla Ala Pro Thr 180 185 190 Tyr Phe Pro Pro His Tyr Phe Val Thr Asn ThrSer Asn Gly Asp Glu 195 200 205 Tyr Glu Phe Asn Leu Val Asp Gly Ala ValAla Thr Val Ala Asp Pro 210 215 220 Ala Leu Leu Ser Ile Ser Val Ala ThrArg Leu Ala Gln Lys Asp Pro 225 230 235 240 Ala Phe Ala Ser Ile Arg SerLeu Asn Tyr Lys Lys Met Leu Leu Leu 245 250 255 Ser Leu Gly Thr Gly ThrThr Ser Glu Phe Asp Lys Thr Tyr Thr Ala 260 265 270 Lys Glu Ala Ala ThrTrp Thr Ala Val His Trp Met Leu Val Ile Gln 275 280 285 Lys Met Thr AspAla Ala Ser Ser Tyr Met Thr Asp Tyr Tyr Leu Ser 290 295 300 Thr Ala PheGln Ala Leu Asp Ser Lys Asn Asn Tyr Leu Arg Val Gln 305 310 315 320 GluAsn Ala Leu Thr Gly Thr Thr Thr Glu Met Asp Asp Ala Ser Glu 325 330 335Ala Asn Met Glu Leu Leu Val Gln Val Gly Glu Asn Leu Leu Lys Lys 340 345350 Pro Val Ser Glu Asp Asn Pro Glu Thr Tyr Glu Glu Ala Leu Lys Arg 355360 365 Phe Ala Lys Leu Leu Ser Asp Arg Lys Lys Leu Arg Ala Asn Lys Ala370 375 380 Ser Tyr 385 287 408 PRT Artificial Synthetic polypeptide 287Met Lys Ser Lys Met Ala Met Leu Leu Leu Leu Phe Cys Val Leu Ser 1 5 1015 Asn Gln Leu Val Ala Ala Phe Ser Thr Gln Ala Lys Ala Ser Lys Asp 20 2530 Gly Asn Leu Val Thr Val Leu Ala Ile Asp Gly Gly Gly Ile Arg Gly 35 4045 Ile Ile Pro Gly Val Ile Leu Lys Gln Leu Glu Ala Thr Leu Gln Arg 50 5560 Trp Asp Ser Ser Ala Arg Leu Ala Glu Tyr Phe Asp Val Val Ala Gly 65 7075 80 Thr Ser Thr Gly Gly Ile Ile Thr Ala Ile Leu Thr Ala Pro Asp Pro 8590 95 Gln Asn Lys Asp Arg Pro Leu Tyr Ala Ala Glu Glu Ile Ile Asp Phe100 105 110 Tyr Ile Glu His Gly Pro Ser Ile Phe Asn Lys Ser Thr Ala CysSer 115 120 125 Leu Pro Gly Ile Phe Cys Pro Lys Tyr Asp Gly Lys Tyr LeuGln Glu 130 135 140 Ile Ile Ser Gln Lys Leu Asn Glu Thr Leu Leu Asp GlnThr Thr Thr 145 150 155 160 Asn Val Val Ile Pro Ser Phe Asp Ile Lys LeuLeu Arg Pro Thr Ile 165 170 175 Phe Ser Thr Phe Lys Leu Glu Glu Val ProGlu Leu Asn Val Lys Leu 180 185 190 Ser Asp Val Cys Met Gly Thr Ser AlaAla Pro Ile Val Phe Pro Pro 195 200 205 Tyr Tyr Phe Lys His Gly Asp ThrGlu Phe Asn Leu Val Asp Gly Ala 210 215 220 Ile Ile Ala Asp Ile Pro AlaPro Val Ala Leu Ser Glu Val Leu Gln 225 230 235 240 Gln Glu Lys Tyr LysAsn Lys Glu Ile Leu Leu Leu Ser Ile Gly Thr 245 250 255 Gly Val Val LysPro Gly Glu Gly Tyr Ser Ala Asn Arg Thr Trp Thr 260 265 270 Ile Phe AspTrp Ser Ser Glu Thr Leu Ile Gly Leu Met Gly His Gly 275 280 285 Thr ArgAla Met Ser Asp Tyr Tyr Val Gly Ser His Phe Lys Ala Leu 290 295 300 GlnPro Gln Asn Asn Tyr Leu Arg Ile Gln Glu Tyr Asp Leu Asp Pro 305 310 315320 Ala Leu Glu Ser Ile Asp Asp Ala Ser Thr Glu Asn Met Glu Asn Leu 325330 335 Glu Lys Val Gly Gln Ser Leu Leu Asn Glu Pro Val Lys Arg Met Asn340 345 350 Leu Asn Thr Phe Val Val Glu Glu Thr Gly Glu Gly Thr Asn AlaGlu 355 360 365 Ala Leu Asp Arg Leu Ala Gln Ile Leu Tyr Glu Glu Lys IleThr Arg 370 375 380 Gly Leu Gly Lys Ile Ser Leu Glu Val Asp Asn Ile AspPro Tyr Thr 385 390 395 400 Glu Arg Val Arg Lys Leu Leu Phe 405 288 410PRT Zea mays 288 Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala Thr ValPro Gln 1 5 10 15 Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu SerIle Asp Gly 20 25 30 Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr Ile Ile AlaTyr Leu Glu 35 40 45 Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg IleAla Asp Tyr 50 55 60 Phe Asp Val Ile Ala Gly Thr Ser Thr Gly Ala Leu LeuAla Ser Met 65 70 75 80 Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu PheAla Ala Lys Asp 85 90 95 Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys IlePhe Pro Gln Lys 100 105 110 Lys Ala Gly Leu Leu Thr Pro Leu Arg Asn LeuLeu Gly Leu Val Arg 115 120 125 Gly Pro Lys Tyr Asp Gly Val Phe Leu HisAsp Lys Ile Lys Ser Leu 130 135 140 Thr His Asp Val Arg Val Ala Asp ThrVal Thr Asn Val Ile Val Pro 145 150 155 160 Ala Phe Asp Val Lys Tyr LeuGln Pro Ile Ile Phe Ser Thr Tyr Glu 165 170 175 Ala Lys Thr Asp Thr LeuLys Asn Ala His Leu Ser Asp Ile Cys Ile 180 185 190 Ser Thr Ser Ala AlaPro Thr Tyr Phe Pro Ala His Phe Phe Lys Thr 195 200 205 Glu Ala Thr AspGly Arg Pro Pro Arg Glu Tyr His Leu Val Asp Gly 210 215 220 Gly Val AlaAla Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr 225 230 235 240 LysGlu Val His Arg Arg Asn Pro Asn Phe Asn Ala Gly Ser Pro Thr 245 250 255Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr Gly Ser Ala Lys 260 265270 Gln Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275280 285 Gln Trp Leu Tyr Asn Gly Gly Phe Thr Pro Ile Ile Asp Ile Phe Ser290 295 300 His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser Ile Leu PheGln 305 310 315 320 Ala Leu His Cys Glu Lys Lys Tyr Leu Arg Ile Gln AspAsp Thr Leu 325 330 335 Thr Gly Asn Ala Ser Ser Val Asp Ile Ala Thr LysGlu Asn Met Glu 340 345 350 Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu LysLys Pro Val Ala Arg 355 360 365 Val Asn Ile Asp Thr Gly Val Tyr Glu SerCys Asp Gly Glu Gly Thr 370 375 380 Asn Ala Gln Ser Leu Ala Asp Phe AlaLys Gln Leu Ser Asp Glu Arg 385 390 395 400 Lys Leu Arg Lys Ser Asn LeuAsn Ser Asn 405 410 289 508 PRT Zea mays 289 Arg Pro Thr Arg Pro Arg HisPro Arg Asn Thr Gln Lys Arg Gly Ala 1 5 10 15 Leu Leu Val Gly Trp IleLeu Phe Ser Leu Ala Ala Ser Pro Val Lys 20 25 30 Phe Gln Thr His Met GlySer Ile Gly Arg Gly Thr Ala Asn Cys Ala 35 40 45 Thr Val Pro Gln Pro ProPro Ser Thr Gly Lys Leu Ile Thr Ile Leu 50 55 60 Ser Ile Asp Gly Gly GlyIle Arg Gly Leu Ile Pro Ala Thr Ile Ile 65 70 75 80 Ala Tyr Leu Glu AlaLys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg 85 90 95 Ile Ala Asp Tyr PheAsp Val Ile Ala Gly Thr Ser Thr Gly Ala Leu 100 105 110 Leu Ala Ser MetLeu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu Phe 115 120 125 Ala Ala LysAsp Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile 130 135 140 Phe ProGln Lys Lys Ala Gly Leu Leu Thr Pro Leu Arg Asn Leu Leu 145 150 155 160Gly Leu Val Arg Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys 165 170175 Ile Lys Ser Leu Thr His Asp Val Arg Val Ala Asp Thr Val Thr Asn 180185 190 Val Ile Val Pro Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile Ile Phe195 200 205 Ser Thr Tyr Glu Ala Lys Thr Asp Ala Leu Lys Asn Ala His LeuSer 210 215 220 Asp Ile Cys Ile Ser Thr Ser Ala Ala Pro Thr Tyr Phe ProAla His 225 230 235 240 Phe Phe Lys Thr Glu Ala Thr Asp Gly Arg Pro ProArg Glu Tyr His 245 250 255 Leu Val Asp Gly Gly Val Ala Ala Asn Asn ProThr Met Val Ala Met 260 265 270 Ser Met Leu Thr Lys Glu Val His Arg ArgAsn Pro Asn Phe Asn Ala 275 280 285 Gly Ser Pro Thr Glu Tyr Thr Asn TyrLeu Ile Ile Ser Val Gly Thr 290 295 300 Gly Ser Ala Lys Gln Ala Glu LysTyr Thr Ala Glu Gln Cys Ala Lys 305 310 315 320 Trp Gly Leu Ile Gln TrpLeu Tyr Asn Gly Gly Phe Thr Pro Ile Ile 325 330 335 Asp Ile Phe Ser HisAla Ser Ser Asp Met Val Asp Ile His Ala Ser 340 345 350 Ile Leu Phe GlnAla Leu His Cys Glu Lys Lys Tyr Leu Arg Ile Gln 355 360 365 Leu Tyr TyrAla Gly Tyr Phe Asp Trp Glu Arg Ile Val Arg Gly His 370 375 380 Arg HisGln Gly Glu His Gly Val Ser Asp Ile Asp Arg Pro Gly Ala 385 390 395 400Ala Gln Glu Ala Ser Gly Glu Ser Glu His Arg His Arg Ala Val Arg 405 410415 Val Leu Arg Arg Gly His Lys Cys Thr Val Ala Ser Leu Arg Gln Ala 420425 430 Thr Leu Arg Ala Gln Ala Thr Gln Glu Gln Ser Gln Leu Gln Leu Ile435 440 445 Asn Thr Ser Leu Ser His Ser Met Cys Ser Phe Arg Arg Phe ThrVal 450 455 460 Ser Tyr Phe Phe Asn Phe Asn Ser Val Cys Val Leu Cys ValLeu Cys 465 470 475 480 Val Tyr Gln Thr Phe Lys Phe Asn Gln Lys Lys LysLys Lys Lys Lys 485 490 495 Lys Lys Lys Lys Lys Lys Lys Lys Lys Arg AlaAla 500 505 290 410 PRT Zea mays 290 Met Gly Ser Ile Gly Arg Gly Thr AlaAsn Cys Ala Thr Val Pro Gln 1 5 10 15 Pro Pro Pro Ser Thr Gly Lys LeuIle Thr Ile Leu Ser Ile Asp Gly 20 25 30 Gly Gly Ile Arg Gly Leu Ile ProAla Thr Ile Ile Ala Tyr Leu Glu 35 40 45 Ala Lys Leu Gln Glu Leu Asp GlyPro Asp Ala Arg Ile Ala Asp Tyr 50 55 60 Phe Asp Val Ile Ala Gly Thr SerThr Gly Ala Leu Leu Ala Ser Met 65 70 75 80 Leu Ala Ala Pro Asp Glu AsnAsn Arg Pro Leu Phe Ala Ala Lys Asp 85 90 95 Leu Thr Thr Phe Tyr Leu GluAsn Gly Pro Lys Ile Phe Pro Gln Lys 100 105 110 Lys Ala Gly Leu Leu ThrPro Leu Arg Asn Leu Leu Gly Leu Val Arg 115 120 125 Gly Pro Lys Tyr AspGly Val Phe Leu His Asp Lys Ile Lys Ser Leu 130 135 140 Thr His Asp ValArg Val Ala Asp Thr Val Thr Asn Val Ile Val Pro 145 150 155 160 Ala PheAsp Val Lys Ser Leu Gln Pro Ile Ile Phe Ser Thr Tyr Glu 165 170 175 AlaLys Thr Asp Thr Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile 180 185 190Ser Thr Ser Ala Ala Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Thr 195 200205 Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His Leu Val Asp Gly 210215 220 Gly Val Ala Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr225 230 235 240 Lys Glu Val His Arg Arg Asn Pro Asn Phe Asn Ala Gly SerPro Thr 245 250 255 Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr GlySer Ala Lys 260 265 270 Gln Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala LysTrp Gly Leu Ile 275 280 285 Gln Trp Leu Tyr Asn Gly Gly Phe Thr Pro IleIle Asp Ile Phe Ser 290 295 300 His Ala Ser Ser Asp Met Val Asp Ile HisAla Ser Ile Leu Phe Gln 305 310 315 320 Ala Leu His Cys Glu Lys Lys TyrLeu Arg Ile Gln Asp Asp Thr Leu 325 330 335 Thr Gly Asn Ala Ser Ser ValAsp Ile Ala Thr Lys Glu Asn Met Glu 340 345 350 Ser Leu Ile Ser Ile GlyGln Glu Leu Leu Asn Lys Pro Val Ala Arg 355 360 365 Val Asn Ile Asp ThrGly Leu Tyr Glu Ser Cys Glu Gly Glu Gly Thr 370 375 380 Asn Ala Gln SerLeu Ala Asp Phe Ala Lys Gln Leu Ser Asp Glu Arg 385 390 395 400 Lys LeuArg Lys Ser Asn Leu Asn Ser Asn 405 410 291 410 PRT Zea mays 291 Met GlySer Ile Gly Arg Gly Thr Ala Asn Cys Ala Thr Val Pro Gln 1 5 10 15 ProPro Pro Ser Thr Gly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly 20 25 30 GlyGly Ile Arg Gly Leu Ile Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 40 45 AlaLys Leu Gln Glu Leu Asp Gly Pro Asp Ala Arg Ile Ala Asp Tyr 50 55 60 PheAsp Val Ile Ala Gly Thr Ser Thr Gly Ala Leu Leu Ala Ser Met 65 70 75 80Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro Leu Phe Ala Ala Lys Asp 85 90 95Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys 100 105110 Lys Ala Gly Leu Leu Thr Pro Leu Arg Asn Leu Leu Gly Leu Val Arg 115120 125 Gly Pro Lys Tyr Asp Gly Val Phe Leu His Asp Lys Ile Lys Ser Leu130 135 140 Thr His Asp Val Arg Val Ala Asp Thr Val Thr Asn Val Ile ValPro 145 150 155 160 Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile Ile Phe SerThr Tyr Glu 165 170 175 Ala Lys Thr Asp Ala Leu Lys Asn Ala His Leu SerAsp Ile Cys Ile 180 185 190 Ser Thr Ser Ala Ala Pro Thr Tyr Phe Pro AlaHis Phe Phe Lys Thr 195 200 205 Glu Ala Thr Asp Gly Arg Pro Pro Arg GluTyr His Leu Val Asp Gly 210 215 220 Gly Val Ala Ala Asn Asn Pro Thr MetVal Ala Met Ser Met Leu Thr 225 230 235 240 Lys Glu Val His Arg Arg AsnPro Asn Phe Asn Ala Gly Ser Pro Thr 245 250 255 Glu Tyr Thr Asn Tyr LeuIle Ile Ser Val Gly Thr Gly Ser Ala Lys 260 265 270 Gln Ala Glu Lys TyrThr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile 275 280 285 Gln Trp Leu TyrAsn Gly Gly Phe Thr Pro Ile Ile Asp Ile Phe Ser 290 295 300 His Ala SerSer Asp Met Val Asp Ile His Ala Ser Ile Leu Phe Gln 305 310 315 320 AlaLeu His Cys Glu Lys Lys Tyr Leu Arg Ile Gln Asp Asp Thr Leu 325 330 335Thr Gly Asn Ala Ser Ser Val Asp Ile Ala Thr Lys Glu Asn Met Glu 340 345350 Ser Leu Ile Ser Ile Gly Gln Glu Leu Leu Lys Lys Pro Val Ala Arg 355360 365 Val Asn Ile Asp Thr Gly Leu Tyr Glu Ser Cys Asp Gly Glu Gly Thr370 375 380 Asn Ala Gln Ser Leu Ala Asp Phe Ala Lys Gln Leu Ser Asp GluArg 385 390 395 400 Lys Leu Arg Lys Ser Asn Leu Asn Ser Asn 405 410 292410 PRT Zea mays 292 Met Gly Ser Ile Gly Arg Gly Thr Ala Asn Cys Ala ThrVal Pro Gln 1 5 10 15 Pro Pro Pro Ser Thr Gly Lys Leu Ile Thr Ile LeuSer Ile Asp Gly 20 25 30 Gly Gly Ile Arg Gly Leu Ile Pro Ala Thr Ile IleAla Tyr Leu Glu 35 40 45 Ala Lys Leu Gln Glu Leu Asp Gly Pro Asp Ala ArgIle Ala Asp Tyr 50 55 60 Phe Asp Val Ile Ala Gly Thr Ser Thr Gly Ala LeuLeu Ala Ser Met 65 70 75 80 Leu Ala Ala Pro Asp Glu Asn Asn Arg Pro LeuPhe Ala Ala Lys Asp 85 90 95 Leu Thr Thr Phe Tyr Leu Glu Asn Gly Pro LysIle Phe Pro Gln Lys 100 105 110 Lys Ala Gly Leu Leu Thr Pro Leu Arg AsnLeu Leu Gly Leu Val Arg 115 120 125 Gly Pro Lys Tyr Asp Gly Val Phe LeuHis Asp Lys Ile Lys Ser Leu 130 135 140 Thr His Asp Val Arg Val Ala AspThr Val Thr Asn Val Ile Val Pro 145 150 155 160 Ala Phe Asp Val Lys SerLeu Gln Pro Ile Ile Phe Ser Thr Tyr Glu 165 170 175 Ala Lys Thr Asp ThrLeu Lys Asn Ala His Leu Ser Asp Ile Cys Ile 180 185 190 Ser Thr Ser AlaAla Pro Thr Tyr Phe Pro Ala His Phe Phe Lys Ile 195 200 205 Glu Ala ThrAsp Gly Arg Pro Pro Arg Glu Tyr His Leu Val Asp Gly 210 215 220 Gly ValAla Ala Asn Asn Pro Thr Met Val Ala Met Ser Met Leu Thr 225 230 235 240Lys Glu Val His Arg Arg Asn Pro Asn Phe Asn Ala Gly Ser Pro Thr 245 250255 Glu Tyr Thr Asn Tyr Leu Ile Ile Ser Val Gly Thr Gly Ser Ala Lys 260265 270 Gln Ala Glu Lys Tyr Thr Ala Glu Gln Cys Ala Lys Trp Gly Leu Ile275 280 285 Gln Trp Leu Tyr Asn Gly Gly Phe Thr Pro Ile Ile Asp Ile PheSer 290 295 300 His Ala Ser Ser Asp Met Val Asp Ile His Ala Ser Ile LeuPhe Gln 305 310 315 320 Ala Leu His Cys Glu Lys Lys Tyr Leu Arg Ile GlnAsp Asp Thr Leu 325 330 335 Thr Gly Asn Ala Ser Ser Val Asp Ile Ala ThrLys Glu Asn Met Glu 340 345 350 Ser Leu Ile Ser Ile Gly Gln Glu Leu LeuAsn Lys Pro Val Ala Arg 355 360 365 Val Asn Ile Asp Thr Gly Leu Tyr GluSer Cys Glu Gly Glu Gly Thr 370 375 380 Asn Ala Gln Ser Leu Ala Asp PheAla Lys Gln Leu Ser Asp Glu Arg 385 390 395 400 Lys Leu Arg Lys Ser AsnLeu Asn Ser Asn 405 410 293 337 PRT Zea mays 293 Met Gly Ser Ile Gly ArgGly Thr Ala Asn Cys Ala Thr Val Pro Gln 1 5 10 15 Pro Pro Pro Ser ThrGly Lys Leu Ile Thr Ile Leu Ser Ile Asp Gly 20 25 30 Gly Gly Ile Arg GlyLeu Ile Pro Ala Thr Ile Ile Ala Tyr Leu Glu 35 40 45 Ala Lys Leu Gln GluLeu Asp Gly Pro Asp Ala Arg Ile Ala Asp Tyr 50 55 60 Phe Asp Val Ile AlaGly Thr Ser Thr Gly Ala Leu Leu Ala Ser Met 65 70 75 80 Leu Ala Ala ProAsp Glu Asn Asn Arg Pro Leu Phe Ala Ala Lys Asp 85 90 95 Leu Thr Thr PheTyr Leu Glu Asn Gly Pro Lys Ile Phe Pro Gln Lys 100 105 110 Lys Ala GlyLeu Leu Thr Pro Leu Arg Asn Leu Leu Gly Leu Val Arg 115 120 125 Gly ProLys Tyr Asp Gly Val Phe Leu His Asp Lys Ile Lys Ser Leu 130 135 140 ThrHis Asp Val Arg Val Ala Asp Thr Val Thr Asn Val Ile Val Pro 145 150 155160 Ala Phe Asp Val Lys Tyr Leu Gln Pro Ile Ile Phe Ser Thr Tyr Glu 165170 175 Ala Lys Thr Asp Ala Leu Lys Asn Ala His Leu Ser Asp Ile Cys Ile180 185 190 Ser Thr Ser Ala Ala Pro Thr Tyr Phe Pro Ala His Phe Phe LysThr 195 200 205 Glu Ala Thr Asp Gly Arg Pro Pro Arg Glu Tyr His Leu ValAsp Gly 210 215 220 Gly Val Ala Ala Asn Asn Pro Thr Met Val Ala Met SerMet Leu Thr 225 230 235 240 Lys Glu Val His Arg Arg Asn Pro Asn Phe AsnAla Gly Ser Pro Thr 245 250 255 Glu Tyr Thr Asn Tyr Leu Ile Ile Ser ValGly Thr Gly Ser Ala Lys 260 265 270 Gln Ala Glu Lys Tyr Thr Ala Glu GlnCys Ala Lys Trp Gly Leu Ile 275 280 285 Gln Trp Leu Tyr Asn Gly Gly PheThr Pro Ile Ile Asp Ile Phe Ser 290 295 300 His Ala Ser Ser Asp Met ValAsp Ile His Ala Ser Ile Leu Phe Gln 305 310 315 320 Ala Leu His Cys GluLys Lys Tyr Leu Arg Ile Gln Leu Tyr Tyr Ala 325 330 335 Gly 294 29 DNAArtificial Synthetic construct 294 gggccatggc gcagttggga gaaatggtg 29295 37 DNA Artificial Synthetic construct 295 aacaaagctt cttattgaggtgcggccgct tgcatgc 37

1-24. (Cancelled)
 25. (Cancelled) 26-61. (Cancelled)
 62. An isolatednucleic acid sequence encoding the protein of SEQ ID NO:
 247. 63. Theisolated nucleic acid sequence of claim 62, comprising SEQ ID NO: 246.64. A recombinant vector operatively linked in the 5′ to 3′ orientation,comprising: a) a promoter that directs transcription of a structuralnucleic acid sequence; b) a nucleic acid sequence encoding the proteinof SEQ ID NO: 247; and c) a 3′ transcription terminator.
 65. Therecombinant vector of claim 64, wherein the vector expresses a proteinexhibiting corn rootworm insect inhibitory activity and acyl lipidhydrolase activity.
 66. A recombinant host cell comprising a nucleicacid sequence encoding the protein of SEQ ID NO:
 247. 67. Therecombinant host cell of claim 66, wherein the protein of SEQ ID NO: 247exhibits corn rootworm insect inhibitory activity and acyl lipidhydrolase activity.
 68. A recombinant plant comprising a nucleic acidsequence encoding the protein of SEQ ID NO:
 247. 69. The recombinantplant of claim 68, wherein the protein of SEQ ID NO: 247 exhibits cornrootworm insect inhibitory activity and acyl lipid hydrolase activity.